Identifying a de novo fetal mutation from a maternal biological sample

ABSTRACT

Systems, methods, and apparatus for determining a fractional concentration of fetal DNA in a biological sample are provided. The biological sample may be enriched for nucleic acid molecules in a target region. A plurality of nucleic acid molecules from the enriched biological sample may be sequenced specific to the target region. One or more first loci may be determined. The fetal genome is heterozygous at each first loci such that the fetal genome has a respective first and second allele at that first loci. The maternal genome is homozygous at each first loci such that the maternal genome. For at least one of the first loci, a first number of counts of the respective first allele and a second number of counts of the respective second allele may be determined. The fractional concentration may be determined based on the first and second numbers.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/002,468, entitled “IDENTIFYING A DE NOVO FETAL MUTATION FROM AMATERNAL BIOLOGICAL SAMPLE,” filed on Jun. 7, 2018, which is acontinuation of U.S. application Ser. No. 13/895,304, entitled“IDENTIFYING A DE NOVO FETAL MUTATION FROM A MATERNAL BIOLOGICALSAMPLE,” filed on May 15, 2013, now U.S. Pat. No. 10,093,976, which is adivisional of U.S. application Ser. No. 12/940,993, entitled “FetalGenomic Analysis From a Maternal Biological Sample,” filed on Nov. 5,2010, now U.S. Pat. No. 8,467,976, which claims priority from and is anon-provisional application of U.S. Provisional Application No.61/381,854, entitled “Fetal Genomic Analysis from a Maternal BiologicalSample,” filed Sep. 10, 2010; U.S. Provisional Application No.61/259,075, entitled “Fetal Genomic Analysis from a Maternal BiologicalSample,” filed Nov. 6, 2009; and U.S. Provisional Application No.61/258,567, entitled “Fetal Genomic Analysis,” filed on Nov. 5, 2009,the entire contents of which are herein incorporated by reference forall purposes.

The present application is also related to U.S. application Ser. No.12/178,181, entitled “Diagnosing Fetal Chromosomal Aneuploidy UsingMassively Parallel Genomic Sequencing,” filed Jul. 23, 2008 (AttorneyDocket No. 016285-005220US); U.S. application Ser. No. 12/614,350,entitled “Diagnosing Fetal Chromosomal Aneuploidy Using GenomicSequencing With Enrichment,” filed Nov. 6, 2009 (Attorney Docket No.016285-005221US); and U.S. application Ser. No. 12/940,992, entitled“Size-Based Genomic Analysis,” filed Nov. 5, 2010 (Attorney Docket No.016285-006610US), the entire contents of which are herein incorporatedby reference for all purposes.

REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS ASCII TEXT FILES VIAEFS-WEB

The Sequence Listing written in file080015-006750US-1331717_SequenceListing.txt created on Jun. 9, 2022,12,114 bytes, machine format IBM-PC, MS-Windows operating system, inaccordance with 37 C.F.R. §§ 1.821- to 1.825, is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND

The present invention relates generally to analyzing a fetal genomebased on a maternal sample, and more particularly to determining all orparts of the fetal genome based on an analysis of genetic fragments inthe maternal sample.

The discovery of cell-free fetal nucleic acids in maternal plasma in1997 has opened up new possibilities for noninvasive prenatal diagnosis(Lo Y M D et al Lancet 1997; 350: 485-487; and U.S. Pat. No. 6,258,540).This technology has been rapidly translated to clinical applications,with the detection of fetal-derived, paternally-inherited genes orsequences, e.g. for fetal sex determination, fetal RhD statusdetermination, and determination of whether the fetus has inherited apaternally-inherited mutation (Amicucci P et al Clin Chem 2000; 46:301-302; Saito H et al Lancet 2000; 356: 1170; and Chiu R W K et alLancet 2002; 360: 998-1000). Recent progress in the field has enabledthe prenatal diagnosis of fetal chromosomal aneuploidies, such astrisomy 21, from maternal plasma nucleic acid analysis (Lo Y M D et alNat Med 2007; 13: 218-223; Tong Y K et al Clin Chem 2006; 52: 2194-2202;US Patent publication 2006/0252071; Lo Y M D et al Proc Natl Acad SciUSA 2007; 104: 13116-13121; Chiu R W K et al Proc Natl Acad Sci USA2008; 105: 20458-20463; Fan H C et al Proc Natl Acad Sci 2008; 105:16266-16271; US Patent publication 2007/0202525; and US Patentpublication 2009/0029377).

Another area of significant recent progress is the use of singlemolecule counting methods, such as digital PCR, for the noninvasiveprenatal diagnosis of single gene diseases in which the mother andfather both carry the same mutation. This has been achieved by relativemutation dosage (RMD) analysis in maternal plasma (US Patent application2009/0087847; Lun F M F et al Proc Natl Acad Sci USA 2008; 105:19920-19925; and Chiu R W K et al. Trends Genet 2009; 25: 324-331).

However, such methods use prior knowledge of possible mutations toanalyze specific parts of a genome, and thus may not identify latent oruncommon mutations or genetic diseases. Therefore, it is desirable toprovide new methods, systems, and apparatus that can identify all orparts of a fetal genome using non-invasive techniques.

BRIEF SUMMARY

Certain embodiments of the present invention can provide methods,systems, and apparatuses for determining at least a portion of thegenome of an unborn fetus of a pregnant female. A genetic map of thewhole genome or for selected genomic region(s) can be constructed of thefetus prenatally using a sample containing fetal and maternal geneticmaterial (e.g. from a blood sample of the pregnant mother). The geneticmap can be of sequences that a fetus has inherited from both of itsfather and mother, or just those of one of the parents. Based on one orseveral of such genetic maps, the risk that the fetus would be sufferingfrom a genetic disease or predisposition to a genetic or other diseasesor a genetic trait can be determined. Other application of embodimentsare also described herein.

In one embodiment, DNA fragments from a maternal sample (containingmaternal and fetal DNA) can be analyzed to identify alleles at certainspecified loci (landmarks). The amount of DNA fragments of therespective alleles at these loci can then be analyzed together todetermine the relative amounts of the haplotypes for these loci andthereby determine which haplotypes have been inherited by the fetus fromthe maternal and/or paternal genomes. By identifying the fetalhaplotypes, the fetal genotype at an individual locus within thecorresponding genomic region including the specified loci can bedetermined. In various embodiments, loci where the parents are aspecific combination of homozygous and heterozygous can be analyzed in amanner to determine regions of the fetal genome. In one implementation,reference haplotypes that are representative of haplotypes common in thepopulation are used along with the analysis of the DNA fragments of thematernal sample to determine the maternal and paternal genomes. Otherembodiments are also provided, such as determining mutations,determining a fractional fetal concentration in a maternal sample, anddetermining a proportion of coverage of a sequencing of the maternalsample.

Other embodiments of the invention are directed to systems, apparatus,and computer readable media associated with methods described herein. Inone embodiment, the computer readable medium contains instructions forreceiving data and analyzing data, but not instructions for directing amachine to create the data (e.g. sequencing nucleic acid molecules). Inanother embodiment, the computer readable medium does containinstructions for directing a machine to create the data. In oneembodiment, a computer program product comprises a computer readablemedium storing a plurality of instructions for controlling a processorto perform an operation for methods described herein. Embodiments arealso directed to computer systems configured to perform the steps of anyof the methods described herein, potentially with different componentsperforming a respective step or a respective group of steps.

Reference to the remaining portions of the specification, including thedrawings and claims, will realize other features and advantages ofembodiments of the present invention. Further features and advantages,as well as the structure and operation of various embodiments of thepresent invention, are described in detail below with respect to theaccompanying drawings. In the drawings, like reference numbers canindicate identical or functionally similar elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of a method 100 of determining at least a portionof the genome of an unborn fetus of a pregnant female according toembodiments of the present invention.

FIG. 2 shows two haplotypes for the father and two haplotypes for themother for a particular segment of their respective genomic codeaccording to embodiments of the present invention.

FIG. 3 shows the two types of SNPs in the parental haplotypes of FIG. 2according to embodiments of the present invention.

FIGS. 4A and 4B show an analysis for determining the fetal haplotypesfor the two types of SNPs according to embodiments of the presentinvention.

FIGS. 5A and 5B show the analysis of comparing relative amounts (e.g.counts) of fragments for each locus and whether a result of thecomparison is to classify a particular haplotype as being inherited ornot according to embodiments of the present invention.

FIG. 6 illustrates the effect of changing the likelihood ratio for SPRTclassification according to embodiments of the present invention.

FIG. 7 is a flowchart of a method 700 of determining at least a portionof the genome of an unborn fetus of a pregnant female inherited from thefather according to embodiments of the present invention.

FIG. 8 is a flowchart of a method 800 for determining at least a portionof the genome of an unborn fetus in a region where the mother and fatherare heterozygous according to embodiments of the present invention.

FIG. 9 shows haplotypes of a father and mother that are bothheterozygous in a particular genomic region according to embodiments ofthe present invention.

FIG. 10 is a flow chart illustrating a method 1000 for determiningfractional concentration of fetal material in a maternal sampleaccording to embodiments of the present invention.

FIG. 11 is a flowchart of a method for determining whether a locus isinformative according to embodiments of the present invention.

FIGS. 12A and 12B show the predicted distribution of the counts forallele T (the less abundant allele in scenarios (a) and (c)) for thethree scenarios with an assumed fractional fetal DNA concentration of20% and 5%, respectively, according to embodiments of the presentinvention.

FIGS. 13A, 13B, and 14 show the predicted distributions for the countsof the less abundant allele for a fractional fetal DNA concentration of20%, each for different total counts of molecules corresponding to a SNPaccording to embodiments of the present invention.

FIGS. 15A and 15B show examples of reference haplotypes, parentalhaplotypes taken from the reference haplotypes, and a resulting fetalhaplotypes according to embodiments of the present invention. Hap A=SEQID NO:1; Hap B=SEQ ID NO:2; Hap c=SEQ ID NO:3.

FIG. 16 is a flowchart of a method 1600 for determining at least part ofa fetal genome when a set of reference haplotypes are known, but theparental haplotypes are not known, according to embodiments of thepresent invention.

FIG. 17 shows an example of determining informative loci from analysisof DNA fragments from a maternal sample according to embodiments of thepresent invention. Hap A=SEQ ID NO:1; Hap B=SEQ ID NO:2; Hap c=SEQ IDNO:3.

FIG. 18 shows the three reference haplotypes (Hap A (SEQ ID NO:1)), HapB (SEQ ID NO:2)) and Hap C (SEQ ID NO:3)) and the paternal alleles.

FIG. 19 shows the determination of the parental haplotype from thepaternal alleles according to embodiments of the present invention. HapA=SEQ ID NO:1; Hap B=SEQ ID NO:2; Hap c=SEQ ID NO:3.

FIG. 20 shows the deduction of the maternal genotypes (SEQ ID NO:4) formthe maternal sample analysis according to embodiments of the presentinvention. Hap A=SEQ ID NO:1; Hap B=SEQ ID NO:2.

FIG. 21 shows an embodiment for determining the maternal haplotypes fromthe maternal genotypes and the reference haplotypes according toembodiments of the present invention. Hap A=SEQ ID NO:1; Hap B=SEQ IDNO:2; Hap c=SEQ ID NO:3; maternal genotypes=SEQ ID NO:4.

FIG. 22 shows the determined maternal haplotypes (Hap A=SEQ ID NO:1; HapB =SEQ ID NO:2) and the paternally inherited haplotype (Hap C; SEQ IDNO:3) according to embodiments of the present invention.

FIG. 23 shows the different types of loci (alpha (A) and beta (B)) forthe maternal haplotypes (Hap A=SEQ ID NO:1; Hap B=SEQ ID NO:2) relativeto the paternal haplotype (SEQ ID NO:3) according to embodiments of thepresent invention.

FIG. 24 is a flowchart illustrating a method 2400 of identifying a denovo mutation in the genome of an unborn fetus of a pregnant female.

FIG. 25A shows the absolute number and the percentages of SNPs showingdifferent genotype combinations for the father, mother and fetus (CVS)according to embodiments of the present invention.

FIG. 25B shows a table listing the alignment statistics of the first 20flow cells.

FIG. 26 is a table showing the fractional concentrations of fetal DNAcalculated for SNPs via two methods according to embodiments of thepresent invention.

FIG. 27A shows a plot illustrating the observed percentage of SNPs inthis subset in which a fetal allele could be seen from the sequencingdata for the first 20 flow cells analyzed, and FIG. 27B shows a plot ofthe coverage vs. the number of reads according to embodiments of thepresent invention.

FIGS. 28A and 28B show plots of the correlation between the coverage ofpaternally-inherited alleles and the number of mappable sequence readsand the number of flow cells sequences, respectively, according toembodiments of the present invention.

FIG. 29A shows the correlation between the false-positive rate and thenumber of flow cells sequenced, and FIG. 29B shows the correlationbetween false-positive rate and the number of flow cells sequencedaccording to embodiments of the present invention.

FIG. 30 shows the coverage of the fetal-specific SNPs for differentnumber of flow cells analyzed according to embodiments of the presentinvention.

FIG. 31 shows the accuracy of Type A analysis when data from 10 flowcells were used according to embodiments of the present invention.

FIG. 32 shows the accuracy of Type B analysis when data from 10 flowcells were used according to embodiments of the present invention.

FIG. 33 shows the accuracy of Type A analysis when the data from 20 flowcells were used according to embodiments of the present invention.

FIG. 34 shows the accuracy of Type B analysis when the data from 20 flowcells were used according to embodiments of the present invention.

FIGS. 35A and 35B show reads with a mutations and with a wildtypesequence at codons 41/42 according to embodiments of the presentinvention. Reads with CD41/42 paternally inherited mutation=SEQ IDNOS:5, 5 and 6-13, respectively. Reads with wildtype sequence inCD41/42=SEQ ID NOS:14, 14, 15, 16, 17, 17, 18, 19, 20, 20, 20, 21, 21,22-28, 29, 29, 30, 30, 30, 31, 32, 32, 33, 34, 35, 35, 36, 36, 37, 38,39, 39, 40-42, 43, 43, 44-46, 47, 47, 47, 48-51, 52 and 52,respectively.

FIG. 36 shows a table of a Type A RHDO analysis, while those of the TypeB RHDO analysis are shown in FIG. 37 according to embodiments of thepresent invention.

FIGS. 38A and 38B show the SPRT classification results for case PW226 asan example.

FIG. 39 shows a table summarizing the RHDO analysis results for the fivecases according to embodiments of the present invention.

FIG. 40 shows a plot of sequencing depth against the number of flowcells sequenced according to embodiments of the present invention.

FIG. 41 shows a plot of the sizes of the fetal and total sequences forthe whole genome, and FIGS. 42A-42C show similar plots individually foreach chromosome according to embodiments of the present invention.

FIG. 43 shows a block diagram of an example computer system 4300 usablewith system and methods according to embodiments of the presentinvention.

DEFINITIONS

The term “biological sample” as used herein refers to any sample that istaken from a subject (e.g., a human, such as a pregnant woman) andcontains one or more nucleic acid molecule(s) of interest.

The term “nucleic acid” or “polynucleotide” refers to a deoxyribonucleicacid (DNA) or ribonucleic acid (RNA) and a polymer thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, small noncoding RNA, micro RNA(miRNA), Piwi-interacting RNA, and short hairpin RNA (shRNA) encoded bya gene or locus.

The term “gene” means the segment of DNA involved in producing apolypeptide chain or transcribed RNA product. It may include regionspreceding and following the coding region (leader and trailer) as wellas intervening sequences (introns) between individual coding segments(exons).

The term “clinically relevant nucleic acid sequence” (also referred toas a target sequence or chromosome) as used herein can refer to apolynucleotide sequence corresponding to a segment of a larger genomicsequence whose potential imbalance is being tested or to the largergenomic sequence itself. One example is the sequence of chromosome 21.Other examples include chromosome 18, 13, X and Y. Yet other examplesinclude mutated genetic sequences or genetic polymorphisms or copynumber variations that a fetus may inherit from one or both of itsparents, or as a de novo mutation in the fetus. In some embodiments,multiple clinically relevant nucleic acid sequences, or equivalentlymultiple makers of the clinically relevant nucleic acid sequence, can beused to provide data for detecting the imbalance. For instance, datafrom five non-consecutive sequences on chromosome 21 can be used in anadditive fashion for the determination of possible chromosomal 21imbalance, effectively reducing the needed sample volume to 1/5.

The term “based on” as used herein means “based at least in part on” andrefers to one value (or result) being used in the determination ofanother value, such as occurs in the relationship of an input of amethod and the output of that method. The term “derive” as used hereinalso refers to the relationship of an input of a method and the outputof that method, such as occurs when the derivation is the calculation ofa formula.

The term “parameter” as used herein means a numerical value thatcharacterizes a quantitative data set and/or a numerical relationshipbetween quantitative data sets. For example, a ratio (or function of aratio) between a first amount of a first nucleic acid sequence and asecond amount of a second nucleic acid sequence is a parameter.

As used herein, the term “locus” or its plural form “loci” is a locationor address of any length of nucleotides (or base pairs) which has avariation across genomes.

The term “sequence imbalance” as used herein means any significantdeviation as defined by at least one cutoff value in a quantity of theclinically relevant nucleic acid sequence from a reference quantity. Asequence imbalance can include chromosome dosage imbalance, allelicimbalance, mutation dosage imbalance, haplotype dosage imbalance, andother similar imbalances. As an example, an allelic or mutation dosageimbalance can occur when a fetus has a different genotype from themother, thereby creating an imbalance at a particular locus in thesample.

The term “chromosomal aneuploidy” as used herein means a variation inthe quantitative amount of a chromosome from that of a diploid genome.The variation may be a gain or a loss. It may involve the whole of onechromosome or a region of a chromosome.

The term “haplotype” as used herein refers to a combination of allelesat multiple loci that are transmitted together on the same chromosome orchromosomal region. A haplotype may refer to as few as one pair of locior to a chromosomal region, or to an entire chromosome. The term“alleles” refers to alternative DNA sequences at the same physicalgenomic locus, which may or may not result in different phenotypictraits. In any particular diploid organism, with two copies of eachchromosome (except the sex chromosomes in a male human subject), thegenotype for each gene comprises the pair of alleles present at thatlocus, which are the same in homozygotes and different in heterozygotes.A population or species of organisms typically includes multiple allelesat each locus among various individuals. A genomic locus where more thanone allele is found in the population is termed a polymorphic site.Allelic variation at a locus is measurable as the number of alleles(i.e., the degree of polymorphism) present, or the proportion ofheterozygotes (i.e., the heterozygosity rate) in the population. As usedherein, the term “polymorphism” refers to any inter-individual variationin the human genome, regardless of its frequency. Examples of suchvariations include, but are not limited to, single nucleotidepolymorphism, simple tandem repeat polymorphisms, insertion-deletionpolymorphisms, mutations (which may be disease causing) and copy numbervariations.

DETAILED DESCRIPTION

A construction of a partial genetic map or complete genomic sequence ofan unborn fetus can be provided based on the haplotypes of polymorphicsequences of its parents. The term “haplotype” as used herein refers toa combination of alleles at multiple loci that are transmitted togetheron the same chromosome or chromosomal region. For example, embodimentscan analyze DNA fragments from a maternal sample (containing maternaland fetal DNA) to identify alleles at certain specified loci(landmarks). The amounts of DNA fragments of the respective alleles atthese loci can then be analyzed together to determine the relativeamounts of the haplotypes for these loci and thereby determine whichhaplotypes have been inherited by the fetus from the maternal and/orpaternal genomes. By identifying the fetal haplotypes, the fetalgenotype at an individual locus within the corresponding genomic regionincluding the specified loci can be determined. In various embodiments,loci where the parents are a specific combination of homozygous andheterozygous can be analyzed in a manner to determine regions of thefetal genome. In one implementation, reference haplotypes that arerepresentative of haplotypes common in the population are used alongwith the analysis of the DNA fragments of the maternal sample todetermine the maternal and paternal genomes.

An example of an application of an embodiment for determining at leastpart of a fetal genome could be for paternity testing by comparing thededuced fetal genotype or haplotype with the genotype or haplotype ofthe alleged father. Another example is to detect one or more de novomutations that the fetus has acquired, or detect meiotic recombinationevents that have occurred during the production of gametes from itsparents. These are the gametes that have fertilized, and the resultingzygote has developed into the fetus.

In addition, some embodiments can also allow the genomic sequence of theunborn fetus to be determined at any desired resolution. For example, incertain applications, embodiments can allow the complete or close tocomplete genomic sequence of the fetus to be determined. In oneembodiment, the resolution of the fetal genomic sequence that can bedetermined is dependent on the resolution of the knowledge of thegenomes of the father and mother, in conjunction with the sequencinginformation from the maternal biological sample containing fetal nucleicacids. In the event that the complete or close to complete genomicsequences of the father and mother are known, the complete or close tocomplete genomic sequence of the unborn fetus could be deduced.

In other embodiments, only the genomic sequences of selected regionswithin the genome are elucidated, e.g., for the prenatal diagnosis ofselected genetic, epigenetic (such as imprinting disorders), orchromosomal disorders. Examples of genetic disorders to which anembodiment can be applied include the hemoglobinopathies (such asbeta-thalassemia, alpha-thalassemia, sickle cell anemia, hemoglobin Edisease), cystic fibrosis, and sex-linked disorders (such as hemophiliaand Duchenne muscular dystrophy). Further examples of mutations that canbe detected using an embodiment can be found from the Online MendelianInheritance in Man (www.ncbi.nlm.nih.gov/omim/getmorbid.cgi).

Some embodiments can also be used to determine a fractionalconcentration of fetal DNA, which may be done without any priorknowledge of the specific genomes of the parents. A similar analysis canalso be used to determine a depth of coverage needed for an accuratedetermination of the fetal genome. Thus, this coverage determination canbe used to estimate how much data needs to be analyzed to obtainaccurate results.

I. INTRODUCTION

When a maternal sample (e.g. plasma or serum) is used as the materialfor elucidating the fetal haplotype, there can be two main challenges. Afirst challenge is that maternal plasma or serum consists of a mixtureof fetal and maternal DNA, with fetal DNA being the minor population. Ithas been determined that fetal DNA represents a mean/medianconcentration of some 5% to 10% of the total DNA in maternal plasma inthe first two trimesters of pregnancy (Lo Y M D et al Am J Hum Genet1998; 62: 768-775; Lun F M F et al Clin Chem 2008; 54: 1664-1672). AsDNA is released by maternal blood cells during the blood clottingprocess, the fractional concentration of fetal DNA in maternal serum canbe even lower than that in maternal plasma. Thus, in some embodiments,maternal plasma is preferred over maternal serum.

A second challenge is that fetal DNA and maternal DNA in maternal plasmaconsist of short fragments (Chan K C A et al Clin Chem 2004; 50: 88-92).Indeed the fetal-derived DNA is generally shorter than thematernal-derived DNA in maternal plasma. Most of the fetal DNA inmaternal plasma is less than 200 bp in length. Using such short plasmaDNA fragments alone, it can be challenging to construct the haplotype ofgenetic polymorphisms over long genomic distances. The above-mentionedchallenges for maternal plasma and serum also apply for the detection offetal DNA in maternal urine (Botezatu I et al Clin Chem 2000; 46:1078-1084). Fetal DNA only represents a minor fraction of the DNA in theurine of a pregnant woman, and fetal DNA in maternal urine also consistsof short DNA fragments.

A. Sequencing and Analyzing of Maternal Sample

An approach that some embodiments have taken to address the firstchallenge is to use a method that allows the quantitative genotyping ofnucleic acids obtained from the maternal biological sample with highprecision. In one embodiment of this approach, the precision is achievedby analysis of a large number (for example, millions or billions) ofnucleic acid molecules. Furthermore, the precision can be enhanced bythe analysis of single nucleic acid molecules or the clonalamplification of single nucleic acid molecules. One embodiment usesmassively parallel DNA sequencing, such as, but not limited to thatperformed by the Illumina Genome Analyzer platform (Bentley D R et al.Nature 2008; 456: 53-59), the Roche 454 platform (Margulies M et al.Nature 2005; 437: 376-380), the ABI SOLiD platform (McKernan K J et al.Genome Res 2009; 19: 1527-1541), the Helicos single molecule sequencingplatform (Harris T D et al. Science 2008; 320: 106-109), real-timesequencing using single polymerase molecules (Science 2009; 323:133-138) and nanopore sequencing (Clarke J et al. Nat Nanotechnol. 2009;4: 265-70). In one embodiment, massively parallel sequencing isperformed on a random subset of nucleic acid molecules in the biologicalsample.

In some embodiments, it can be beneficial to obtain as long a sequenceread from each molecule as is possible. One limitation of the length ofthe sequencing reads that can be achieved is the nature of the nucleicacid molecules in the maternal biological sample. For example, it isknown that most DNA molecules in maternal plasma consist of shortfragments (Chan K C A et al Clin Chem 2004; 50: 88-92). Furthermore, theread length has to be balanced against the fidelity of the sequencingsystem at long read lengths. For some of the above-mentioned systems, itmight be preferable to obtain sequences from both ends of the molecule,the so-called paired-end sequencing. As an illustration, one approach isto perform 50 bp of sequencing from each end of a DNA molecule, thusresulting in a total of 100 bp of sequence per molecule. In anotherembodiment, 75 bp of sequencing from each end of a DNA molecule, thusresulting in a total of 150 bp of sequence per molecule, can be done.

After the sequencing is performed, the sequences are then aligned backto a reference human genome. As embodiments elucidate the genomicvariations inherited by an unborn fetus from its parents, the alignmentalgorithm can be able to cope with sequence variations. One example ofsuch a software package is the Efficient Large-Scale Alignment ofNucleotide Databases (ELAND) software produced by Illumina. Anotherexample of such a software package is the SOAP (short oligonucleotidealignment program) and SOAP2 software (Li R et al. Bioinformatics 2008;24:713-714; Li R et al. Bioinformatics 2009; 25:1966-1967).

The amount of DNA sequencing that may need to be performed can depend onthe resolution at which the fetal genetic map or fetal genomic sequencemay need to be constructed. In general, the more molecules that aresequenced the higher the resolution. Another determinant of theresolution of the fetal genetic map or fetal genomic sequence at a givenlevel, or depth, of DNA sequencing is the fractional concentration offetal DNA in the maternal biological sample. In general, the higher thefractional fetal DNA concentration, the higher is the resolution of thefetal genetic map or fetal genomic sequence that can be elucidated at agiven level of DNA sequencing. As the fractional concentration of fetalDNA in maternal plasma is higher than that in maternal serum, maternalplasma is a more preferred maternal biological sample type than maternalserum for some embodiments.

The throughput of the above-mentioned sequencing-based methods can beincreased with the use of indexing or barcoding. Thus, a sample orpatient-specific index or barcode can be added to nucleic acid fragmentsin a particular nucleic acid sequencing library. Then, a number of suchlibraries, each with a sample or patient-specific index or barcode, aremixed together and sequenced together. Following the sequencingreactions, the sequencing data can be harvested from each sample orpatient based on the barcode or index. This strategy can increase thethroughput and thus the cost-effectiveness of embodiments of the currentinvention.

In one embodiment, the nucleic acid molecules in the biological samplecan be selected or fractionated prior to quantitative genotyping (e.g.sequencing). In one variant, the nucleic acid molecules are treated witha device (e.g. a microarray) which can preferentially bind nucleic acidmolecules from selected loci in the genome (e.g. the region onchromosome 7 containing the CFTR gene). Then the sequencing can beperformed preferentially on nucleic acid molecules captured by thedevice. This scheme will allow one to target the sequencing towards thegenomic region of interest. In one embodiment of this scheme a Nimblegensequence capture system (www.nimblegen.com/products/seqcap/index.html)or an Agilent SureSelect Target Enrichment System(www.opengenomics.com/SureSelect_Target_Enrichment_System), or similarplatforms, can be used. In some embodiments, the nucleic acid moleculesfrom the selected regions of the genome are subjected to randomsequencing.

In another embodiment, the genomic region of interest in the biologicalsample can be first amplified by one set or multiple set ofamplification primers. Then, the quantitative genotyping, for example,sequencing, can be performed on the amplified products. In oneimplementation of this scheme, the RainDance(www.raindancetech.com/technology/per-genomics-research.asp) system canbe used. In some embodiments, the amplified nucleic acid molecules aresubjected to random sequencing.

A size fractionation step can also be performed on the nucleic acidmolecules in the biological sample. As fetal DNA is known to be shorterthan maternal DNA in maternal plasma (Li et al Clin Chem 2004; 50:1002-1011; US Patent Application 20050164241; US Patent application20070202525), the fraction of smaller molecular size can be harvestedand then used for the quantitative genotyping, for example, sequencing.Such a fraction would contain a higher fractional concentration of fetalDNA than in the original biological sample. Thus, the sequencing of afraction enriched in fetal DNA can allow one to construct the fetalgenetic map or deduce the fetal genomic sequence with a higherresolution at a particular level of analysis (e.g. depth of sequencing),than if a non-enriched sample has been used. This can therefore make thetechnology more cost-effective. As examples of methods for sizefractionation, one could use (i) gel electrophoresis followed by theextraction of nucleic acid molecules from specific gel fractions; (ii)nucleic acid binding matrix with differential affinity for nucleic acidmolecules of different sizes; or (iii) filtration systems withdifferential retention for nucleic acid molecules of different sizes.

In yet another embodiment, one could preferentially analyze nucleic acidmolecules of a specific size or size range following the nucleic acidsequencing. For example, one could perform paired-end sequencing inwhich both ends of a DNA molecule are sequenced. Then, the genomiccoordinates of both of these ends could be mapped back to a referencehuman genome. Then one could deduce the size of the molecule bysubtracting the genomic coordinates of both ends. One way to performsuch paired-end sequencing is to use the paired-end sequencing protocolof the Illumina Genome Analyzer. Another method to deduce the size of aDNA molecule is to sequence the entire DNA molecule. This is mostreadily done by sequencing platforms with relatively long read lengths,such as the Roche 454 platform (Marguelis et al Nature 2005;437:376-380) and the Pacific Biosciences single molecule, real-time(SMRT™) technology (Eid et al Science 2009; 323: 133-138). Following thededuction of the size of the nucleic acid molecules, one could choose tofocus the subsequent analysis on molecules of less than a particularsize cutoff, thereby enriching in the fractional concentration of fetalDNA. Analysis of this subset of molecules can allow the fetal geneticmap or fetal genomic sequences to be deduced with fewer analyzedmolecules after the size selection than be if this procedure has notbeen done. In one embodiment, a size cutoff of 300 bp is used. In yetother embodiments, a size cutoff of 250 bp, 200 bp, 180 bp, 150 bp, 125bp, 100 bp, or 75 bp could be used.

B. Using Parental Genomes as Scaffolds

To address the second challenge, some embodiments can use haplotypes ofthe chromosomes of the mother as a ‘scaffold’. The haplotypes of thechromosomes of the father can also be used as another ‘scaffold’. Thisscaffold can be compared against genetic information of the fetusobtained from the maternal sample containing fetal DNA. This fetalgenetic information can be used to determine how the scaffold of themother and/or father have been erected in the fetal genome, therebyusing the component parts of the scaffold to determine the resultingfetal genome.

The parental haplotypes can be constructed from genomic DNA from thefather and mother, and from other members of the family, e.g. a siblingof the fetus in the current pregnancy. It is possible that theavailability of the parental haplotypes can become increasinglycommonplace, in view of the reduction in the costs of genomicsequencing. In one scenario, if one or both parents already have theirgenomes sequenced and their haplotypes on one or more chromosomalregions have been determined, then this information can be used as theabove-mentioned scaffold.

Any genotyping platform known to those of skill in the art that caninterrogate sequence variations in the genome can be used, including DNAsequencing, microarrays, hybridization probes, fluorescence-basedtechniques, optical techniques, molecular barcodes and single moleculeimaging (Geiss G K et al. Nat Biotechnol 2008; 26: 317-325), singlemolecule analysis, PCR, digital PCR, mass spectrometry (such as theSequenom MassARRAY platform), etc. As a more extreme example, the DNAsequence of the father and mother can be determined by whole genome DNAsequencing using a massively parallel sequencing method (e.g. Bentley DR et al. Nature 2008; 456: 53-59; McKernan K J et al. Genome Res 2009;19: 1527-1541). An example of sequence variations that may be ofinterest are single nucleotide polymorphisms (SNPs). A particularlypreferred method for determining the parental genotypes is by microarrayanalysis of SNPs on a genomewide scale, or at selected genomic regions,e.g. those containing genes whose mutations can cause genetic diseases(such as genes in the beta-globin cluster, or the cystic fibrosistransmembrane conductance regulator (CFTR) gene). Apart from sequencevariations, copy number variations can also be used. Sequence variationsand copy number variations are both referred to as polymorphic geneticfeatures (PMF).

In one aspect, the maternal genotypes on the chromosomes or chromosomalregions of interest can be constructed into haplotypes. One way in whichthis can be performed is by the analysis of other family members relatedto the mother, e.g. a son or daughter of the mother, a parent, asibling, etc. Another way in which the haplotypes can be constructed isthrough other methods well known to those skilled in the art mentionedabove.

The genotype information can then be extended into haplotype informationof the parents by comparison with the genotype information from otherfamily members, for example, a sibling of the fetus of the currentpregnancy, or from the genotypes of the grandparents, etc. Haplotypes ofthe parents can also be constructed by other methods well known to thoseskilled in the art. Examples of such methods include methods based onsingle molecule analysis such as digital PCR (Ding C and Cantor C R.Proc Natl Acad Sci USA 2003; 100: 7449-7453; Ruano Get al. Proc NatlAcad Sci USA 1990; 87: 6296-6300), sperm haplotyping (Lien S et al. CurrProtoc Hum Genet 2002; Chapter 1:Unit 1.6) and imaging techniques (XiaoM et al. Hum Mutat 2007; 28: 913-921). Other methods include those basedon allele-specific PCR (Michalatos-Beloin S et al. Nucleic Acids Res1996; 24: 4841-4843; Lo Y M D et al. Nucleic Acids Res 1991; NucleicAcids Res 19: 3561-3567), cloning and restriction enzyme digestion(Smirnova A S et al. Immunogenetics 2007; 59: 93-8), etc. Yet othermethods are based on the distribution and linkage disequilibirumstructure of haplotype blocks in the population which allow the maternalhaplotype to be inferred from statistical assessments (Clark A G. MolBiol Evol 1990; 7:111-22; 10:13-9; Salem R M et al. Hum Genomics 2005;2:39-66).

C. Using Genomic Information of Maternal Sample to Assemble the Scaffold

In one embodiment, to work out which of the maternal chromosomes havebeen passed onto the fetus, a relative haplotype dosage (RHDO) method isused. A general principle of this approach is as follows for an exampleof where the mother is heterozygous for each of the geneticpolymorphisms. Thus, there are two haplotypes, and the relative dosageof these haplotypes would be 1:1. However, in the maternal sample, thepresence of a small proportion of fetal DNA might alter the relativehaplotype dosage. This is because the fetus would have inherited half ofits haplotype complement from the mother and the other half from thefather. Furthermore, for each chromosome, the fetus might have inheriteda ‘patchwork’ of haplotypes which have originated from one or the otherhomologous chromosomes from each parent, depending on the occurrence ofmeiotic recombination. All of these factors might deviate the relativehaplotype dosage from the 1:1 ratio in the maternal constitutional DNA.Thus, for a given chromosome or chromosomal region, the constituentalleles of these haplotypes can be sought from analytic data (e.g.sequencing data) generated from the maternal sample.

Then, a statistical procedure can be performed to determine the relativehaplotype dosage, or if one of these haplotypes is overrepresented overthe other haplotype. The classification threshold for this statisticalprocedure can be adjusted depending on the fractional fetal DNAconcentration. In general, a higher fractional fetal DNA concentrationcan allow the threshold to be reached with fewer molecules. Theclassification threshold can also be adjusted depending on the number ofsuccessfully classified fragments that one wishes to achieve across thegenome or the genomic regions of interest. In one embodiment, thesequential probability ratio test (SPRT) can be used.

In one embodiment, a relative mutation dosage (RMD), as described in USPatent application 2009/0087847) can be used to determine a relativeamount of an allele at particular polymorphisms of the mother. Theserelative amounts can be used in determining a haplotype of the fetus(e.g. when the polymorphisms are at consecutive or linked loci). In oneimplementation of this targeted approach is the use of the polymerasechain reaction (PCR) to amplify specific sequences from selected partsof the genome for RMD analysis. To extend this RMD approach to determinefetal inheritance over a large genomic region or the whole genome, alarge volume of maternal sample is needed.

In an embodiment using random sequencing, the genomic regions ofinterest are not specifically targeted. Thus, the number of sequencesobtained in the genomic regions of interest may not be as numerous as ina targeted approach (unless very deep sequencing is performed). However,counts can be pooled the counts across a number of linked polymorphisms,to achieve the necessary statistical power for diagnostic purposes. Apractical implication of using this sequencing embodiment is that it cansave costs by avoiding the need for excessively deep sequencing. It alsorequires an input of a lesser amount of maternal sample than digital PCRbased approaches.

Furthermore, it can be desirable to perform such RHDO analysis inblocks. In other words, each chromosome can be analyzed in one, orpreferably more than one block. In one aspect, the latter can allowmeiotic recombination to be observed. For example, a haplotype of asegment of a particular chromosome of the fetus can appear to have comefrom one of the maternal homologous chromosomes, while another segmentof the same fetal chromosome appears to possess the haplotype from theother maternal homologous chromosome. An SPRT analysis can allow thissegmentation to be performed.

For example, SPRT analysis can be performed on neighboring SNPsdemonstrating the required parental genotype configuration (i.e. thefather being homozygous and the mother being heterozygous) starting fromone end of a chromosome. This will continue until the SPRT analysis hasindicated that one of the maternal haplotype is predominant in thematernal plasma analytic data (e.g. sequencing data). Then, the SPRTanalysis can be ‘reset’ and start afresh from the next neighboring SNPdemonstrating the required parental genotype configuration. This canagain continue until the SPRT analysis has once again indicated that oneof the maternal haplotype is predominant in the maternal plasma analyticdata (e.g. sequencing data). This process can continue until the lastselected SNP on the said chromosome. Then, these various SPRT-determinedhaplotype segments on the chromosome can be compared with the haplotypesof the two homologous chromosomes in the mother's genome. A meioticrecombination is seen when the haplotype segments in the fetus appear tohave switched from one maternal homologous chromosome to another one.This system can also work even if there is more than one meioticrecombination per chromosome.

As is described later, RHDO analysis can also be carried out for genomicregions in which the father and mother are both heterozygous for theconstituent genetic polymorphisms. This scenario is particularly usefulfor situation when the father and mother share a mutant copy of thedisease gene from the same ancestral origin, such as when they areconsanguineous, or when the predominant mutation for the disease is dueto a large founder effect (i.e. most individuals with the mutation hasinherited the same haplotype from a common ancestral founder of thepopulation). Thus, the haplotypes of the father and mother in thisregion can be used to deduce the fetal haplotype.

II. CONSTRUCTING FETAL GENOME FROM MATERNAL GENOME

Constructing a fetal genetic map or elucidating the fetal genomicsequence with explicit knowledge of the parental genomes is nowdescribed.

A. Method

FIG. 1 is a flowchart of a method 100 of determining at least a portionof the genome of an unborn fetus of a pregnant female. The fetus has afather and a mother being the pregnant female. The father has a paternalgenome with two haplotypes and the mother has a maternal genome with twohaplotypes. Method 100 analyzes nucleic acid molecules (fragments) froma biological sample obtained from the pregnant female to determine thegenome of the fetus. Method 100 is described primarily for the exampleof when the father is homozygous and the mother is heterozygous at aplurality of loci, while other examples describe other embodiments

Method 100 and any of the methods described herein may be totally orpartially performed with a computer system including a processor, whichcan be configured to perform the steps. Thus, embodiments are directedto computer systems configured to perform the steps of any of themethods described herein, potentially with different componentsperforming a respective step or a respective group of steps. Althoughpresented as numbered steps, steps of methods herein can be performed ata same time or in a different order. Additionally, portions of thesesteps may be used with portions of other steps from other methods. Also,all or portions of a step may be optional. Additionally, any of thesteps of any of the methods can be performed with modules, circuits, orother means for performing these steps.

In step 110, a first plurality of loci are identified at which thematernal genome is heterozygous. In one embodiment, this determinationcan be performed at part of a genotyping of the father and mother at thegenomewide level or at selected genomic loci of interest. In otherembodiments, the determination of the first plurality of loci can bemade during an analysis of the maternal sample, which is described inlater sections.

In step 120, each of the two maternal haplotypes covering the firstplurality of loci are determined. As mentioned above, the maternalgenome could be obtained from direct sequencing. In other embodiments,genotyping can be done at a plurality of loci and then use a mappedgenome of someone that is expected to have a similar genome, e.g. from afamily member or from a reference genome that is common in a same orsimilar population. In one embodiment, step 120 can be performed firstfor all or parts of the maternal genome and then the maternal genome canbe investigated to find loci where the mother is heterozygous.

In one aspect, it is not essential to construct the haplotypes of thechromosomes of the father. However, if the paternal haplotypes could beconstructed then additional information could be obtained from thesequencing results. One such additional information includes the factthat relative haplotype dosage analysis can be performed for regions forwhich both parents are heterozygous. Another additional piece ofinformation which can be obtained if the paternal haplotype is availableis information concerning meiotic recombination involving one or morepaternal chromosomes, and to determine if disease alleles linked to suchpolymorphisms have been passed onto to the fetus.

In step 130, an allele inherited by the fetus from the father at each ofthe first plurality of loci is determined. Some embodiments use genomicloci which are homozygous for the father, but heterozygous for themother (as mentioned in step 110). Thus, if the father is homozygous atthe loci, then the allele that is inherited from the father is known.The genotyping of the father to determine loci at which the father ishomozygous can be determined in any of the ways described herein. In oneembodiment, the determination of the first plurality of loci can bedetermined based on the genotyping of the father and mother in order tofind loci at which the father is homozygous and at which the mother isheterozygous.

In another embodiment, a second plurality of loci of the paternal genomethat are heterozygous can be used to determine the paternal haplotypeinherited by the fetus at the first plurality of loci at which thefather is homozygous. For example, if the maternal genome is homozygousat the second plurality of loci, alleles that are present in thepaternal genome at respective ones of the second plurality of loci andabsent in the maternal genome can be identified. The inherited paternalhaplotype can then be identified as the haplotype with the identifiedalleles, and used to determine the allele inherited from the father atthe first plurality of loci. These aspects of determining a paternalhaplotype are discussed in more detail below.

In step 140, a plurality of nucleic acid molecules from a biologicalsample obtained from the pregnant female analyzed. The sample contains amixture of maternal and fetal nucleic acids. The maternal biologicalsample can be taken and then received for analysis. In one embodiment,maternal plasma and serum is used. In other embodiments, maternal blood,maternal urine, maternal saliva, uterine lavage fluid, or fetal cellsobtained from maternal blood can be used.

In one embodiment, analyzing a nucleic acid molecule includesidentifying a location of the nucleic acid molecule in the human genome,and determining an allele of the nucleic acid molecule at the individuallocus. Thus, one embodiment can perform quantitative genotyping usingthe determined alleles of the nucleic acid molecules from the samelocus. Any method that will allow the determination of the genomiclocation and allele (information as to genotype) of nucleic acidmolecules in the maternal biological sample can be used. Some of suchmethods are described in U.S. applications Ser. No. 12/178,181 and Ser.No. 12/614350, and application entitled “Size-Based Genomic Analysis.”

In step 150, based on the determined alleles of the nucleic acidmolecules, amounts of respective alleles at each of the first pluralityof loci are determined. In one embodiment, the amounts can be the numberof alleles of each type at a first locus. For example, six A and four T.In another embodiment, an amount can be a size distribution of thenucleic acid molecules having a particular allele. For example, arelative amount can also include a size distribution of the fragmentswith a particular genotype, which can convey a relative amount offragments at certain lengths. Such relative amounts can also provideinformation as to which genotype is in the fetal genome, since fetalfragments tend to be smaller than the maternal fragments. Some examplesof amounts and methods are described in U.S. applications Ser. No.12/178,181 and Ser. No. 12/614350, and application entitled “Size-BasedGenomic Analysis.”

In one embodiment, the relative amounts of the alleles at a locus canprovide information as to which genotype was inherited by the fetus(e.g. after a dataset has reached sufficient statistical power). Forexample, the relative amounts can be used to determine whether asequence imbalance occurs relative to the mother's genotypes at a locus.The related patent applications cited above provide examples ofembodiments for detecting a sequence imbalance at a particular locus orregion.

In step 160, relative amounts of the respective alleles of the nucleicacid molecules at more than one locus of the first plurality of loci arecompared. In some embodiments, amounts of each allele at each locus ofthe first plurality of loci comprising the haplotypes are aggregatedbefore making a comparison. The aggregated amounts of the parentalhaplotypes can then compared to determine if a haplotype isover-represented, equally represented or under-represented. In otherembodiments, the amounts for the alleles at a locus are compared, andcomparisons at multiple loci are used. For example, a separation value(e.g. a difference or a ratio) can be aggregated, which can be used in acomparison with a cutoff value. Each of these embodiments can apply toany of the comparisons steps described herein.

In various embodiments, the relative amounts can be a count of a numberof each fragment with a particular allele at a particular locus, a countof a number of fragments from any locus (or any loci in a region) on aparticular haplotype, and a statistical value of the count (e.g., anaverage) at a particular locus or on a particular haplotype. Thus, inone embodiment, the comparison can be a determination of a separationvalue (e.g. a difference or a ratio) of one allele vs. another allele ateach loci.

In step 170, based on the comparison, the haplotype that is inherited bythe unborn fetus from the mother at the portion of the genome covered bythe first plurality of loci can be determined. In one embodiment, towork out which of the maternal chromosomes have been passed onto thefetus, a relative haplotype dosage (RHDO) method is used, e.g., asmentioned above. As the mother is heterozygous for each of the firstloci, the first loci correspond to two haplotypes for the genomic regionof first loci. The relative dosage of these haplotypes would be 1:1 ifthe sample was just from the mother. Deviations or lack of deviationsfrom this ratio can be used to determine the haplotype of the fetus thatis inherited from the mother (and the father, which is addressed in moredetail later). Thus, for a given chromosome or chromosomal region, theconstituent alleles of these haplotypes can be sought from the analyticdata (e.g. sequencing data) generated in step 130.

Since a plurality of loci are analyzed and compared to the haplotype ofthe mother, the sequences between the loci can be attributed to aparticular haplotype. In one embodiment, if several loci match aparticular haplotype, then the sequence segments between the loci can beassumed to be the same as that of the maternal haplotype. Because of theoccurrence of meiotic recombination, the final haplotype inherited bythe fetus can consist of a patchwork of ‘haplotype segments’ originatingfrom one of these two homologous chromosomes. Embodiments can detectsuch recombination.

The resolution in which one could detect such recombination is dependenton the number and distribution of the genetic markers that one hasdetermined in the father's and mother's constitutional DNA, and thethreshold that one uses in the subsequent bioinformatic analysis (usingfor example the SPRT). For example, if the comparison suggests that theallele inherited from the mother at each of a first set of consecutiveloci correspond to the first haplotype, then the first haplotype isdetermined to be inherited for the genomic location corresponding to thefirst set of loci. If a second set of consecutive loci suggest that thesecond haplotype is inherited, then the second haplotype is determinedto be inherited for the genomic location corresponding to the second setof loci.

In one embodiment, as a plurality of loci are analyzed, the haplotypecan be determined with greater accuracy. For example, the statisticaldata for one loci may not be determinative, but when combined with thestatistical data of other loci, a determination of which haplotype isinherited can be made. In another embodiment, each loci can be analyzedindependently to make a classification, and then the classifications canbe analyzed to provide a determination of which haplotype is inheritedfor a given region.

In one embodiment, a statistical procedure can be performed to determinethe relative haplotype dosage (e.g. if one of these haplotypes isoverrepresented over the other haplotype). The classification thresholdfor this statistical procedure can be adjusted depending on thefractional fetal DNA concentration. In general, a higher fractionalfetal DNA concentration can allow the threshold to be reached with fewermolecules. The classification threshold can also be adjusted dependingon the number of successfully classified segments that one wishes toachieve across the genome or the genomic regions of interest.

Referring back to FIG. 1, in step 180, the fetal genome can be analyzedfor mutations. For example, embodiments can be used to search for apanel of mutations causing genetic diseases in a particular population.Examples of mutations that can be detected using embodiments can befound from the Online Mendelian Inheritance in Man(www.ncbi.nlm.nih.gov/omim/getmorbid.cgi). These mutations can besearched for during steps 140-160; or as a separate step as describedhere. For example, in families in which the father is a carrier of oneor more mutations which are absent in the mother, then the mutation(s)could be searched for from the analytic data (e.g. sequencing data) fromthe maternal biological sample.

Apart from detecting the actual mutation, one could also look forpolymorphic genetic markers which are linked to the mutant or wildtypeallele in the father or mother. For example, RHDO analysis may revealthat the fetus has inherited the haplotype from the mother that is knownto carry a mutation for a disease. Embodiments of the invention can alsobe used for the noninvasive prenatal diagnosis of diseases caused bydeletions of chromosomal regions, e.g. the Southeast Asian deletioncausing alpha-thalassemia. In the scenario in which both the father andthe mother are carriers of the deletion, if the fetus is homozygous forthe deletion, and if massively parallel sequencing is performed onmaternal plasma DNA, then there should be a reduction in the frequenciesof DNA sequences originating from the deleted region in maternal plasma.

B. Example

This section describes an example of embodiments (e.g. of method 100)applied to single-nucleotide polymorphism (SNPs) in which the mother isheterozygous. The SNP alleles on the same chromosome form a haplotype,with the mother having a homologous pair of each chromosome, and thustwo haplotypes. To illustrate how such a determination is performed,consider a segment on chromosome 3, e.g., as shown in FIG. 2.

FIG. 2 shows two haplotypes for the father and two haplotypes for themother for a particular segment of their respective genomic code. FiveSNPs were found within this segment in which the father and mother werehomozygous and heterozygous, respectively, for all 5 of these SNPs. Thetwo homologous chromosomes of the father possessed the same haplotype(Hap), i.e., A-G-A-A-G (from top to bottom in FIG. 2). For simplicity,the paternal haplotypes are called Hap I and Hap II, bearing in mindthat both of these are identical for this set of 5 SNPs. For the mother,two haplotypes were observed, namely Hap III, A-A-A-G-G and Hap IV,G-G-G-A-A.

The SNPs in this example could be further classified into two types.FIG. 3 shows the two types of SNPs according to embodiments of thepresent invention. Type A consists of those SNPs in which the paternalalleles were the same as those on the maternal haplotype III. Type Bconsists of those SNPs in which the paternal alleles were the same asthose on the maternal haplotype IV.

These two types of SNPs can require slightly different mathematicalhandling. Thus, in the Type A scenario, the fetal inheritance ofhaplotype III would result in the overrepresentation of haplotype III,relative to haplotype IV, in maternal plasma (FIG. 4A). For example,looking at just one SNP 410 for ease of discussion, the allele A isinherited from the father, and if Hap III is inherited from the mother,then the fetus will be contributing two A alleles to the sample, whichwill cause an overrepresentation of A. If the fetus had inheritedhaplotype IV then no overrepresentation would be seen, since the fetuswould also be heterozygous with A and G at the locus.

On the other hand, in the Type B scenario, the fetal inheritance ofhaplotype III would result in the equal representation of haplotype IIIand haplotype IV in maternal plasma (FIG. 4B). For example, lookingagainst SNP 420, the inheritance of G from the father and A as part ofHap III would cause the fetus to contribute equal amounts of A and G atSNP 420, just like the mother. If the fetus had inherited haplotype IV,then overrepresentation would be observed as is evident from thediscussion above.

FIGS. 5A and 5B shows the analysis of comparing relative amounts (e.g.counts) of fragments for each locus and whether a result of thecomparison is to classify a particular haplotype as being inherited ornot. Any genomic location in which there is a SNP which fits one ofthese genotype configurations of the father and mother (e.g. Type A orType B scenarios) can be used for this example. From the maternal plasmasequencing data, one can focus on the number of sequenced moleculescorresponding to a particular allele of the SNP. An SPRT analysis (orother comparison method) can be used to determine if there was anyallelic imbalance between these alleles (Lo Y M D et al Proc Natl AcadSci USA 2007; 104: 13116-13121).

FIG. 5A shows an analysis for type A SNPs. As shown, for each SNP, aSPRT comparison of the relative amounts (e.g. as defined by a separationvalue) to a cutoff value provides a classification. In one embodiment,if the classification threshold for SPRT was reached then the fetalinheritance of a particular maternal haplotype was concluded. Countingfor the SPRT analysis can then be reset. Then, an analysis can move ontoa neighboring SNP fitting the required genotype configuration, eitherfrom the telomeric-to-centromeric direction, or vice versa; and the newSPRT analysis can begin with this next SNP.

On the other hand, in one embodiment, if the classification for SPRT wasnot reached with the SNP, then we can also move onto a neighboring SNPin a similar fashion, except that the counts for the next SNP can beadded to the previous one and then SPRT can again be performed. Thisprocess can continue until the classification threshold had beenreached. FIG. 5A and FIG. 5B illustrate the operation of this processfor Type A and Type B analyses. In one embodiment, the classificationsare analyzed together to make a total classification for a region. Forexample, if a classification is obtained for a first group of SNPs andfor the next group of SNPs, the classification of the two can becompared to see if the classification is consistent.

FIG. 6 illustrates the effect of changing the likelihood ratio for SPRTclassification (Zhou W et al. Nat Biotechnol 2001;19:78-81; Karoui N Eet al. Statist Med 2006;25:3124-33). In general a lower likelihood ratiofor classification, e.g., 8, can allow classification to be made moreeasily. This can result in a larger number of classified regions withinthe genome. However, a number of such regions can be expected to bemisclassified. On the other hand, a higher likelihood forclassification, e.g., 1200, can only allow classification when more SNPshave been scored. This can result in a smaller number of classifiedregions within the genome. The number and proportion of misclassifiedregions can be expected to be lower when compared with situations when alower classification threshold was used.

In one embodiment, a classification is made only if two consecutive SPRTclassifications result in the same haplotype (referred to as the “twoconsecutive blocks” algorithm). In one aspect, the “two consecutiveblocks” algorithm can increase the accuracy of classification. In someembodiments, for any stretch of sequence, an embodiment can firstperform an SPRT analysis for Type A SNPs, and then do another SPRTanalysis for the Type B SNPs. In one embodiment, one can consider thescenario for a stretch of sequence for which the Type A and Type B SNPsform two interlacing groups of genetic landmarks (e.g. SNPs). Inembodiments using the “two consecutive blocks” algorithm, the two blockscan be of different types.

The SPRT results from the Type A and Type B analyses can allow one tocheck for concordance or discordance in their classification results. Toenhance the classification accuracy, one embodiment (“interlacingapproach”) could only make a classification if both the Type A and TypeB analyses for a given genomic region can yield consistent results. Ifthe two analyses yield discordant results, we can look at theclassification results of the two contiguous regions of classificationnext to the region, one at the centromeric end and the other one at thetelomeric end. If these two contiguous regions yield concordant results,then we can classify the first region as a continuous haplotype withthese two regions. If these two contiguous regions do not yieldconcordant results, then we can move onto the next two contiguousregions until concordance is seen. One variant of this theme is to movein just one direction and to take the classification results of the nextone, or two, or even more contiguous regions as the results of theoriginal region concerned. The general principle is to use theclassification results of adjacent genomic regions to confirm theclassification results of a particular region.

III. DETERMINATION OF THE PATERNAL ALLELES INHERITED BY THE FETUS

FIG. 7 is a flowchart of a method 700 of determining at least a portionof the genome of an unborn fetus of a pregnant female inherited from thefather. Method 700 analyzes nucleic acid molecules (fragments) from abiological sample obtained from the pregnant female to determine thegenome of the fetus. The sample contains a mixture of maternal and fetalnucleic acids.

In step 710, each of a plurality of nucleic acid molecules from thebiological sample are analyzed to identify a location of the nucleicacid molecule in the human genome, and determine an allele type of thenucleic acid molecule. Thus, genotypes of the nucleic acid molecules ata particular location (locus) can be determined in one embodiment. Anyof the methods described above and elsewhere may be used for thisanalysis.

In step 720, a first plurality of loci are determined at which thepaternal genome is heterozygous and the maternal genome is homozygous.In one embodiment, the first plurality of loci are obtained bydetermining the paternal and maternal genomes. The genomes can be minedfor genomic loci in which the father is heterozygous and the mother ishomozygous.

In step 730, the haplotype that is inherited by the unborn fetus fromthe father at the portion of the genome covered by the first pluralityof loci is determined based on the determined genotypes at the firstplurality of loci. In one embodiment, the allele of each of these lociwhich is possessed by the father, but absent in the genome of themother, is sought for in the analytic data (e.g. sequencing data). Thecombination of these alleles would indicate the haplotypes of thechromosomes that the fetus has inherited from the father.

In another embodiment, if the haplotypes of each of the chromosomes orthe chromosomal regions of interest in the father's genome is known,then one can also determine where meiotic recombination has occurredduring spermatogenesis in the father. Hence, paternal meioticrecombination is seen when the haplotype of a stretch of DNA in apaternally-inherited chromosome differs between the fetus and thefather. The inclusion of such recombination information can be usefulwhen the analytic data (e.g. sequencing data) are used for the prenataldiagnosis of a genetic disease by linkage analysis to geneticpolymorphisms.

IV. FATHER AND MOTHER ARE HETEROZYGOUS FOR A GENOMIC REGION

Embodiments can address a scenario in which the father and mother areheterozygous for a genomic region. This scenario can be particularlyrelevant in families in which the father and mother are consanguineous.When a disease is associated with a predominant mutation which hasresulted from a large founder effect can also be relevant. In suchcircumstances, it is to be expected that if the father and mother of theunborn fetus are both carriers of the mutant gene, then the haplotype ofthe chromosome carrying the mutant copy of the gene can essentially beidentical, except for the occurrence of meiotic recombination events.This type of analysis can be especially useful for autosomal recessivediseases such as cystic fibrosis, beta-thalassemia, sickle cell anema,and hemoglobin E disease.

FIG. 8 is a flowchart of a method 800 for determining at least a portionof the genome of an unborn fetus in a region where the mother and fatherare heterozygous according to embodiments of the present invention.

In step 810, a first plurality of loci are determined at which thefather and mother are both heterozygous. In one embodiment, the firstloci can be determined by any of the methods mentioned herein. Forexample, all or regions of the parental genomes can be sequenced, ordifferent parts genotyped to find the first loci. Thus, each of the twopaternal and each of the two maternal haplotypes at the first pluralityof loci can be known.

As an example, FIG. 9 shows haplotypes of a father and mother that areboth heterozygous in a particular genomic region. As shown, both parentshave a mutant gene (allele) in region 1. Specifically, Hap I of thefather and Hap III of the mother have the mutant gene. Also as shown,the father and mother can each have the other copy of the chromosomecarrying the wildtype copy of the gene. Specifically, Hap II of thefather and Hap IV of the mother have the wildtype gene. Thus, thisexample has relevance in determining whether a fetus has inherited amutant gene. The chromosomes from the father and mother that carry thewildtype gene have an identical haplotype in the immediate vicinity ofthe gene, but might have divergent haplotypes further away from thegene. As this chromosome would likely have a diverse ancestral origin,this chromosome would unlikely have identical haplotypes between thefather and mother throughout the whole chromosome.

In step 820, a second plurality of loci are determined at which thefather is heterozygous, but at which the mother is homozygous. As shown,the first and second pluralities of loci are on the same chromosome.Region 2 shows such second loci. Region 2 can be chosen such that thefather is heterozygous for one or more SNPs in this region while themother is homozygous in this region.

In step 830, fragments from a sample of the pregnant female can beanalyzed to identify a location in the human genome and a genotype. Thelocation can be used to determine whether a fragment (nucleic acidmolecule) includes one or more of the first loci or one or more of thesecond loci. This information can then be used to determine thehaplotype inherited from the father and the haplotype inherited from themother.

In step 840, which of the two paternal haplotypes has been inherited bythe fetus is determined by analyzing the determined genotypes of theplurality of nucleic acid molecules from the biological sample at atleast one of the second loci. For example, the SNP alleles which areuniquely present in the father's genome, but absent in the mother'sgenome, such as the T allele marked by * and the A allele marker by ⁺ inFIG. 9, can be sought for from the analytic data (e.g. location andgenotype resulting from step 710) of the maternal biological sample. Ascan be done for method 700, if the T allele marked by * is detected frommaternal plasma, then it means that haplotype II (Hap II) is inheritedby the fetus from the father. Conversely, if the A allele marked by ⁺ isdetected from maternal plasma, then it means that Hap I is inherited bythe fetus from the father.

In step 850, comparing relative amounts of the determined genotypes ofnucleic acid molecules at more than one of the first plurality of loci.In one embodiment, amounts at each locus are aggregated and the relativeamounts of the maternal haplotypes are compared. The relative amountscan refer to counted numbers, size distributions, and any otherparameter that can convey information as to which genotype is in thefetal genome at a particular locus.

In step 860, based on the paternal haplotype determined to be inheritedby the fetus and based on the comparison of the relative amounts,determining the haplotype that is inherited by the unborn fetus from themother at the portion of the genome covered by the first plurality ofloci. Thus, an RHDO analysis (e.g. as described above) of SNPs in Region1 from the analytic data of the maternal biological sample can becarried out to determine which one of the two maternal haplotypes hasbeen inherited by the fetus, taking the paternal haplotype inherited bythe fetus in Region 2 into consideration. In one embodiment, it isassumed that there is no recombination between Regions 1 and 2 whenthese regions are passed from the parents to the fetus.

For example, consider the scenario when the fetus has been determined tohave inherited Hap I from the father through Region 2 analysis. Then,the fetal inheritance of Hap III (which is identical to Hap I atRegion 1) from the mother will result in the overrepresentation of HapIII relative to Hap IV in maternal plasma. Conversely, if the fetus hasinherited Hap IV from the mother, then equal representation of Hap IIIand Hap IV will be observed in maternal plasma.

As another example, consider the scenario when the fetus has beendetermined to have inherited Hap II from the father through Region 2analysis. Then, the fetal inheritance of Hap IV (which is identical toHap II at Region 1) from the mother will result in theoverrepresentation of Hap IV relative to Hap III in maternal plasma.Conversely, if the fetus has inherited Hap III from the mother, thenequal representation of Hap III and Hap IV will be observed in maternalplasma.

In the previous sections, we have deduced the fetal genome and thefractional fetal DNA concentration using the data obtained from thesequencing of the maternal plasma DNA, as well as the genotypeinformation of the parents of the fetus. In the following sections, wedescribe embodiments for deducing the fractional fetal DNA concentrationand fetal genotype without prior information of the maternal andpaternal genotypes/haplotypes.

V. DETERMINATION OF FRACTIONAL FETAL DNA CONCENTRATION

In some embodiments, an optional step is to determine a fractional fetalDNA concentration. In various aspects, this fractional concentration canguide the amount of analysis (e.g. amount of sequencing required) orallow one to estimate the accuracy of the analysis for a given amount ofdata (e.g. depth of genome sequence coverage). The determination of thefractional fetal DNA concentration can also be useful for determining acutoff to determine a classification of which haplotype and/or genotypeare inherited.

In one embodiment, the fractional fetal DNA concentration can bedetermined by mining the analytic data (e.g. as can be obtained in step140 and 710) for loci that are homozygous for the father and for themother, but with different alleles. For example, for a SNP with twoalleles, A and G; the father can be AA and the mother can be GG, andvice versa. For such loci, the fetus would be an obligate heterozygote.In the example above, the fetal genotype would be AG, and a proportionof allele A in the maternal sample can be used to determine thefractional fetal DNA concentration. In another embodiment, a statisticalanalysis can be made to determine a locus where the mother is homozygousand the fetus is heterozygous. In this manner, no prior informationabout the mother's genome or the paternal genome is needed.

As alternatives to mining the analytic data, the fractional fetal DNAconcentration can also be determined by another approach, such as theuse of PCR assays, digital PCR assays or assays based on massspectrometry, on a panel of polymorphic genetic markers (Lun F M F et alClin Chem 2008; 54: 1664-1672). Another alternative is to use one ormore genomic loci which exhibit different DNA methylation between thefetus and mother (Poon L L M et al. Clin Chem 2002; 48: 35-41; Chan K CA et al. Clin Chem 2006; 52: 2211-2218; U.S. Pat. No. 6,927,028). As yetanother alternative is to use an approximate fractional fetal DNAconcentration determined from a reference population, e.g. at a similargestational age. However, as the fractional fetal DNA concentrationcould vary from sample to sample, this latter approach may be expectedto be less precise than if the concentration is measured specificallyfor the sample being tested.

A. Determining Fractional Concentration for Obligate Heterozygote

In embodiments where the fetus is an obligate heterozygote, one candetermine the fractional fetal DNA concentration using the followingseries of calculations (e.g. using massively parallel sequencing). Let pbe the counts of the fetal allele that is absent from the maternalgenome. Let q be the counts of the other allele, i.e. the allele that isshared by the maternal and fetal genomes. Fractional fetal DNAconcentration is given by the following equation:

$\frac{2p}{p + q}$

In one implementation, this calculation can be performed on thecumulative data across different polymorphic genetic loci or polymorphicgenetic features that fulfill the parental genotype configuration (e.g.,both parents being homozygous, but for different alleles).

B. Determination Based on Informative SNPs

The fractional concentration of fetal DNA can also be determined for anylocus at which the mother is homozygous and the fetus is heterozygous,and not just when the mother is homozygous for one allele and the fatheris homozygous for a different allele. Both methods provide whether alocus is informative. The term “informative SNP” can be used indifferent contexts depending on what information is desired. In onecontext, the information is an allele in the fetal genome at aparticular locus that is not present in the maternal genome at thatlocus. Thus, the subset of SNPs that the mother is homozygous and thefetus is heterozygous can be referred to as “informative SNPs” for thecontext of determining fetal DNA concentration. Instances where themother and fetus are both heterozygous, but for at least one differentallele, can also be used as an informative SNP. However, triallelic SNPsare relatively uncommon in the genome.

FIG. 10 is a flow chart illustrating a method 1000 for determiningfractional concentration of fetal material in a maternal sampleaccording to embodiments of the present invention. In step 1010,fragments from a sample of the pregnant female can be analyzed toidentify a location in the human genome and an allele type (which canlead to a genotype determination at the location). In one embodiment,the fragments are analyzed by sequencing a plurality of nucleic acidmolecules from the biological sample obtained from the pregnant female.In other embodiments, real-time PCR or digital PCR can be used.

In step 1020, one or more first loci are determined to be informative.In some embodiments, the maternal genome is homozygous, but anon-maternal allele is detected in the sample at an informative locus.In one embodiment, the fetal genome is heterozygous at each first lociand the maternal genome is homozygous at each first loci. For example,the fetal genome can have a respective first and second allele (e.g. TA)at a first locus, and the maternal genome can have two of the respectivesecond allele (e.g. AA) at the first locus. However, such loci may notbe a priori known, e.g., in situations where the fetus is not anobligate heterozygote.

In one embodiment to determine an informative locus, the SNPs at whichthe mother is homozygous are considered. For SNPs that the mother ishomozygous, the fetus is either homozygous for the same allele or isheterozygous. For example, if a SNP is polymorphic for A and T, and themother has a genotype of AA, the genotype of the fetus is either AA orTA. In this case, the presence of the T allele in the maternal plasmasample would indicate the fetal genotype is TA instead of AA. Certainembodiments can address how much of a presence of the T allele indicatesa genotype of TA by calculating a necessary cutoff, as is describedbelow.

In step 1030, for at least one of the first loci, a first number p ofcounts of the respective first allele and a second number q of counts ofthe respective second allele are determined. In one embodiment, thecounts of the fetal-specific (the T allele) and the shared (the Aallele) alleles in maternal plasma can be determined by a variety ofmethods, for example, but not limited to real-time PCR, digital PCR, andmassively parallel sequencing.

In step 1040, the fractional concentration is calculated based on thefirst and second numbers. In one embodiment, in a pregnant woman withgenotype AA and the genotype of her fetus being TA, the fractional fetalDNA concentration (f) can be calculated using the equation: f=2×p/(p+q),where p represents the counts for the fetal-specific allele (allele T)and q represents the counts for the allele shared by the mother and thefetus (allele A).

In another embodiment, by the use of multiple informative SNPs, thefractional concentration of fetal DNA in maternal plasma can beestimated with increased accuracy. For the use of the allele counts ofmultiple SNPs (a total of n SNPs), the fractional concentration of fetalDNA (f) can be calculated using the equation

$f = \frac{\sum_{i = 1}^{n}{2p_{i}}}{\sum_{i = 1}^{n}\left( {p_{i} + q_{i}} \right)}$

where p_(i) represents the counts for the fetal-specific allele for theinformative SNP_(i); q_(i) represents the counts for the allele sharedby the mother and the fetus for the informative SNP_(i); and nrepresents the total number of informative SNPs. The use of the allelecounts of multiple SNPs can increase the accuracy of the estimation ofthe fractional fetal DNA concentration.

C. Fractional Concentration Without Explicit Genetic Information OfParents

A method for determining the fractional fetal DNA concentration in amaternal plasma sample which does not require prior informationregarding the genotypes of the fetus and the mother is now described. Inone embodiment, the identification of informative SNPs is made from thecounts of different alleles at these SNP loci in maternal plasma. Thus,method 1000 can be used, along with the determination of the informativeSNPs based on embodiments described below. First, a description ofprobabilities is provided to help understand a calculation of a cutoffthat is used to identify informative SNPs.

In one embodiment, the probability of detecting the fetal-specificallele follows the Poisson distribution. The probability (P) ofdetecting the fetal-specific allele can be calculated using thefollowing equation: P=1−exp (−f×N/2), where f represents the fractionalconcentration of fetal DNA in the maternal plasma sample, N representsthe total number of molecules corresponding to this particular SNP locusbeing analyzed; and exp( ) represents the exponential function. In oneaspect, P can be considered an expected distribution as it is not adistribution resulting from measuring an amount of molecules across manysamples. In other embodiments, other distributions can be used.

Assuming that the fractional concentration of fetal DNA is 5% (a typicalvalue for the first trimester pregnancy) and 100 molecules (maternal+fetal) corresponding to this SNP locus are analyzed (equivalent to theamount contained in 50 diploid genomes), the probability of detectingthe fetal-specific allele (the T allele) is 1−exp(−0.05×100/2)=0.92. Theprobability of detecting the fetal-specific allele would increase withthe fractional fetal DNA concentration and the number of molecules beinganalyzed for the SNP locus. For example, if the fetal DNA concentrationis 10% and 100 molecules are analyzed, the probability of detecting thefetal-specific allele is 0.99.

Therefore, at a SNP locus for which the mother is homozygous, thepresence of an allele different from the maternal one in maternal plasmacan indicate that the SNP is “informative” for the calculation of thefractional fetal DNA concentration. The probability of missing anyinformative SNP can be dependent on the number of molecules analyzed. Inother words, for any desired confidence of detecting the informativeSNPs, the number of molecules that needs to be analyzed to obtain adesired accuracy can be calculated according to the Poisson probabilityfunction.

Using the above analysis, some embodiments can determine if a locus isinformative or not when the genotype of the mother is not known. In oneembodiment, loci at which two different alleles are detected in thematernal plasma sample are identified. For example, for a SNP locus withtwo possible alleles A and T, both the A and the T alleles are detectedin the maternal plasma.

FIG. 11 is a flowchart of a method 1100 for determining whether a locusis informative according to embodiments of the present invention. In oneembodiment, method 1100 can be used to implement step 1020 of method1000. In another embodiment, one step of method 1100 is to determine acutoff value based on a statistical distribution, and another uses thecutoff value to determine whether a locus (SNP) is informative.

In step 1110, a cutoff value is determined for a number of predictedcounts of the respective first allele at the specific locus. In oneimplementation, the cutoff value predicts whether the maternal genome ishomozygous and the fetal genome is heterozygous. In one embodiment, thecutoff value is determined based on a statistical distribution ofnumbers of counts for different combinations of homozygosity andheterozygosity at the specific locus. For example, an allelic frequencydistribution can be predicted using the Poisson distribution function.

In step 1120, based on an analysis of the nucleic acid molecules of thematernal sample (e.g. from step 1010), a first allele and a secondallele are detected at the locus. For example, a set of fragments couldbe mapped to the locus being analyzed and the first allele or the secondallele was detected. The first allele can correspond to one of therespective first alleles from step 1020, and the second allele cancorrespond to one of the respective second alleles. In one embodiment,if two different alleles are not detected, then it is known that thelocus is not informative.

In step 1130, a number of actual counts of the respective first alleleat the locus is determined based on the analysis of the nucleic acidmolecules. For example, sequencing results of the plurality of nucleicacid molecules can be counted to determine the number of times afragment having a genotype of the first allele is mapped to the locus.

In step 1140, the locus is identified as one of the first loci based ona comparison of the number of actual counts to the cutoff value. In oneaspect, a cutoff value can be used to differentiate between threepossibilities: (a) the mother is homozygous (AA) and the fetus isheterozygous (AT); (b) the mother is heterozygous (AT) and the fetus isheterozygous (AT); and (c) the mother is heterozygous (AT) and the fetusis homozygous for (AA) or (TT). For the sake of illustration, theexamples below assume the fetal genotype to be AA in scenario (c).However, the calculation would be the same if the genotype of the fetusis TT. An informative locus would have the possibility (a).

In one embodiment, the locus is identified as one of the first loci whenthe number of actual counts is less than the cutoff value. In anotherembodiment, a lower threshold can also be used to ensure that a spuriousmapping did not occur.

Embodiment for determining the cutoff is now described. Based on thephysiologically possible fractional fetal DNA concentration (thisinformation is available from previous studies) and the total number ofmolecules corresponding to the SNP locus, the distribution of theallelic counts can be predicted for the three possible scenarios above.Based on the predicted distribution, a cutoff value can be determinedfor interpreting the observed allelic counts in maternal plasma todetermine if a SNP is “informative” (i.e. scenario (a)) or not.

The fractional concentration of fetal DNA typically ranges from 5% to20% in early pregnancy and ranges from 10% to 35% in late pregnancy (Lunet al., Microfluidics digital PCR reveals a higher than expectedfraction of fetal DNA in maternal plasma. Clin Chem 2008; 54:1664-72).Thus, in one embodiment, the predicted distributions of the alleliccounts for 5% and 20% fractional concentration of fetal DNA weredetermined.

FIG. 12A shows the predicted distribution of the counts for allele T(the less abundant allele in scenarios (a) and (c)) for the threescenarios with an assumed fractional fetal DNA concentration of 20%.FIG. 12B shows the predicted distribution of the counts for allele T(the less abundant allele for scenarios (a) and (c)) for the threescenarios with the assumption of 5% fetal DNA. In both predicted models,a total of 200 molecules were assumed to be analyzed for the SNP locus.

Using the presence of 40 counts of the less abundant allele (the Tallele) as a cutoff, the three possibilities can be statisticallydiscriminated. In other words, for any SNP locus with two allelesdetected in the maternal plasma and with a total of 200 molecules beinganalyzed, if the allelic frequency of the minor allele (the lessabundant allele) is less than 40, the SNP locus can be regarded as“informative”. For fractional fetal DNA concentrations of 5% and 20%,the differentiation of “informative” SNPs (scenario (a)) from the SNPsfor which the mother is heterozygous (scenarios (b) and (c)) would be100% accurate.

In practice, the total number of molecules detected can be different fordifferent SNPs. For each SNP locus, a specific predicted distributioncurve can be constructed by taking into account the total number ofmolecules detected in the maternal plasma sample covering the SNP locus.In other words, the count cutoff for determining whether a SNP isinformative or not can vary among SNPs and depends on the number oftimes the SNP locus has been counted.

The following table shows the allele counts of three SNP loci inmaternal plasma for a maternal plasma sample that was sequenced. Foreach of the three SNPs, two different alleles are detected in thematernal plasma sample. The total numbers of counts detected in maternalplasma corresponding to these three SNPs are different.

Total no. SNP locus SNP id Allele (counts) Allele (counts) of counts 1.rs3107146 A (10) G (163) 173 2. rs7522344 G (9) T (112) 121 3. rs2272908A (72) G (62) 134

The predicted distributions for the counts of the less abundant allelefor a fractional fetal DNA concentration of 20% and different totalcounts of molecules corresponding to a SNP are shown in FIGS. 13A, 13B,and 14. The predicted distributions were drawn using an assumed fetalDNA concentration of 20% because this represents the higher limit offetal DNA concentration in the first trimester. The higher the fetal DNAconcentration, the more overlap is expected between the distributioncurves of the minor allele for which the mother is homozygous for themajor allele against that when the mother is heterozygous. Thus, it ismore specific to derive cutoffs for the minor allele counts using ahigher fetal DNA concentration for the prediction of informative SNPs.

FIG. 13A shows a predicted distribution for the counts of the lessabundant allele with a total number of 173 molecules and fractionalfetal DNA concentration of 20%. In one embodiment, based on thisdistribution, a cutoff criterion of less than 40 for the counts of theless abundant allele can be suitable for identifying the informativeSNPs. As the counts for the A allele is 10, the SNP locus no. 1 isregarded as “informative” for the calculation of the fractional fetalDNA concentration.

FIG. 13B shows a predicted distribution for the counts of the lessabundant allele with a total number of 121 molecules and fractionalfetal DNA concentration of 20%. In one embodiment, based on thisdistribution, a cutoff value of less than 26 for the counts of the lessabundant allele can be suitable for identifying the informative SNPs. Asthe number of counts for the T allele is 9, the SNP locus no. 2 isregarded as “informative” for the calculation of the fractional fetalDNA concentration.

FIG. 12 shows a predicted distribution for the counts of the lessabundant allele with a total number of 134 molecules and fractionalfetal DNA concentration of 20%. In one embodiment, based on thisdistribution, a cutoff value of less than 25 for the counts of the lessabundant allele can be suitable for identifying the informative SNPs. Asthe number of counts for the T allele is 62, the SNP locus no. 3 isregarded as “not informative” and would not be used for the calculationof fetal DNA fractional concentration.

In some embodiments, using the equation f=2×p/(p+q), the fractionalconcentration of fetal DNA can be calculated using the allele counts forSNP 1 and 2 and combined. The results are shown below.

Calculation based on SNP locus Fractional concentration of fetal DNA 1.10 × 2/(10 + 163) = 11.6% 2. 9 × 2/(9 + 112) = 14.9% 1. and 2. (10 + 9)× 2/(10 + 9 + 163 + 112) = 12.9%

D. Determination of Depth Coverage of the Fetal Genome

Besides obtaining a fractional concentration, embodiments can determinea percentage coverage of the fetal genome that the analytic procedure(e.g. sequencing) in step 1010 has accomplished. In some embodiments,informative loci can be used to determine the percentage of coverage.For example, any of the examples from above can be used. In oneembodiment, loci at which the fetus is an obligate heterozygote can beused. In another embodiment, loci at which the fetus is determined to beheterozygous and the mother is homozygous may be used (e.g. using method1100).

The fragments that have been mapped to the informative loci can be usedto determine a proportion of coverage. In one embodiment, a proportionof loci of the first plurality of loci in which a respective firstallele is detected from the sequencing results is determined. Forexample, if the fetus is TA at a locus and the mother is AA at thelocus, then the allele T should be detected in the sequencing results ifthat locus has been sequenced. Thus, the proportion of the fetal genomethat has been sequenced from the biological sample can be calculatedbased on this proportion. In one embodiment, the proportion of the firstloci where the fetal-specific allele is seen can be taken as thepercentage coverage of the fetal genome. In other embodiments, theproportion can be modified based on where the loci are at. For example,a percentage coverage can be determined for each chromosome. As anotherexample, the percentage can be estimated at less than the proportion ifthe first loci do not form a good representation of the genome. Asanother example, a range might be provided where the proportion is oneend of the range. While a high percentage, i.e. approaching 100%,signifies close to complete coverage of the fetal genome, most geneticdiseases can be diagnosed with much less than 100% coverage, e.g. 80%,or 50%, or less.

VI. NO PRIOR INFORMATION OF MATERNAL AND PATERNAL GENOME

In previous sections, some embodiments have determined a genetic map ofa fetus (or a portion of a fetal genome) when the haplotypes of themother and the genotypes of the father are known. Other embodiments havedemonstrated that fractional fetal DNA concentration can be determinedby analyzing the maternal plasma DNA without prior knowledge about thegenotypes of the mother, the father, or the fetus. In yet otherembodiments, we now further describe a method for determining thegenetic map of a fetus (or a portion of a fetal genome) using RHDOanalysis without prior information of the maternal and paternalgenotypes/haplotype(s).

In one embodiment, the information of reference (e.g. common or known)haplotypes of the population in which the parents belong to is used.This information can be used for deducing the maternal and paternalhaplotypes. An example is used to illustrate the principle of thismethod. Information concerning such reference haplotypes can beobtained, for example, from the website of the International HapMapProject (hapmap.ncbi.nlm.nih.gov/).

As part of an illustrative example, assume that three referencehaplotypes (Hap A, Hap B and Hap C as shown in FIG. 15A) are present inthe population. Each of these three haplotypes consists of 14 SNP lociand, for each locus, there are two possible alleles. In this example,the father possesses Hap B and Hap C whereas the mother possesses Hap Aand Hap B, as shown in FIG. 15B. This example assumes that the fetusinherits Hap A from the mother and Hap C from the father. Therefore, thefetus possesses Hap A and Hap C, as shown in FIG. 15B.

FIG. 16 is a flowchart of a method 1600 for determining at least part ofa fetal genome when a set of reference haplotypes are known, but theparental haplotypes are not known, according to embodiments of thepresent invention.

In step 1610, the maternal sample can be analyzed to identify SNPs atwhich the mother is homozygous and the fetus is heterozygous. Thisanalysis can be done in a similar fashion as a determination of whethera locus is informative, as described above. Thus, in one embodiment,methods 1000 and/or 1100 can be used. In other embodiments describedabove, the maternal and paternal genomes can be analyzed to determineinformation to perform the fetal genome mapping.

FIG. 17 shows an example of determining informative loci from analysisof DNA fragments from a maternal sample. For each of the 14 loci, thecounts of the two alleles for each locus are determined. The counts ofthese alleles can be determined, for example but not limited to, usingreal-time PCR, digital PCR, and massively parallel sequencing. For eachof these loci, two different alleles would be detected in the maternalplasma. In contrast to those SNPs at which the mother is heterozygous,the proportion of the two alleles would be significantly different. Thefetus-specific allele (the allele that the fetus inherits from thefather) would be much less abundant compared with the maternal allele.The informative loci 1710 are marked in FIG. 17.

In step 1620, one or more alleles of the paternal haplotype inherited bythe fetus are deduced. In one embodiment, each of the loci 1710 can beused to determine the inherited parternal haplotype. For example, thepaternal allele that the fetus has inherited can be identified as thefetal-specific allele for loci 1720 because the fetal-specific allele isthe allele is much less abundant than the maternal allele in thematernal sample.

In step 1630, the paternal alleles are compared to the referencehaplotypes to determine the haplotype inherited from the father. Incertain embodiments, a number of possible fetal haplotypes can bededuced, each with its own probability. One or more of the most likelyfetal haplotypes can then be used for subsequent analysis, or forclinical diagnosis.

In the example shown in FIG. 18, there are three possible haplotypes(Hap A, Hap B and Hap C) in the population. From the maternal plasmaanalysis, four SNPs have been identified as being homozygous for themother and heterozygous for the fetus, thus representing the paternalalleles that the fetus inherits. The genotypes at these four SNPs fitthe pattern of Hap C. Therefore, the fetus has inherited Hap C from thefather, as shown in FIG. 19. In other words, for all the SNPs within thesame haplotype block, the paternal alleles that the fetus has inheritedcan be deduced.

In step 1640, loci (e.g. SNPs) at which the mother is heterozygous canbe determined. In one embodiment, analysis of the maternal sample canprovide SNPs that the mother is heterozygous. For example, at each ofthese SNPs, two different alleles can be detected in maternal plasma. Incontrast to the SNPs that the mother is homozygous and the fetus isheterozygous which the fetal-specific allele only contributes a smallproportion of the total alleles in maternal plasma, the counts of thetwo alleles would be similar for SNPs where the mother is heterozygous.Thus, the complete maternal genotype for all the SNP loci within thehaplotype block could be determined from maternal plasma analysis, e.g.,as shown in FIG. 20.

In step 1650, the maternal haplotypes are deduced from the maternalgenotypes from step 1640 by comparing the genotypes at the loci to thehaplotype information of the relevant population. FIG. 21 shows anembodiment for determining the maternal haplotypes from the maternalgenotypes and the reference haplotypes. In the example being used, themother is homozygous for the G allele at the third SNP locus. As onlyHap A and Hap B fulfill this criterion, this indicates the mother hasone of the three haplotype combinations, namely Hap A/HapA, Hap A/Hap Bor Hap B/HapB. In addition, as the mother is heterozygous for A and Cfor the first SNP, we can deduce the mother has the haplotypecombination of Hap A/Hap B. In one embodiment, more than one possibilitymight result, and each possibility could be tested in the next step.From the above analyses, the haplotypes of the mother and the haplotypethat the fetus inherits from the father have been determined. FIG. 22shows the determined maternal haplotypes and the paternally inheritedhaplotype.

In step 1660, the maternal haplotype inherited by the fetus isdetermined from the maternal haplotypes identified in step 1650 and thepaternally inherited haplotype identified in step 1630. Using thisinformation, an embodiment can use RHDO analysis to determine whichmaternal haplotype is passed onto the fetus. An RHDO analysis can beperformed according to any of the embodiment described herein.

In one embodiment, for the RHDO analysis, the SNPs at which the motheris heterozygous can be divided into two types, namely type alpha andtype beta (e.g. as shown in FIG. 23 and as described above). Type alphaSNPs refer to those loci where the paternal allele passed onto the fetusis identical to the maternal allele located on Hap A. For type alphaSNPs, if the fetus inherits Hap A from the mother, the allele on Hap Awould be overrepresented in maternal plasma. On the other hand, if thefetus inherits Hap B from the mother, the two maternal alleles would beequally represented in maternal plasma.

Type beta SNPs refer to those loci where the paternal allele passed ontothe fetus is identical to the maternal allele located on Hap B. For typebeta SNPs, if the fetus inherits Hap B from the mother, the allele onHap B would be overrepresented in maternal plasma. However, if the fetusinherits Hap A from the mother, the two maternal alleles would beequally represented in maternal plasma. The potential overrepresentationof Hap A or Hap B alleles can be determined using RHDO analysis.

In some embodiments, to apply RHDO analysis on a particular regionwithout prior information of the maternal haplotypes and paternalgenotypes, a relatively high-fold coverage of the SNPs within thehaplotype block can be required, for example, 200 moleculescorresponding to a SNP locus may need to be analyzed in one embodiment.This information can be obtained by, for example but not limited to,real-time PCR, digital PCR and massively parallel sequencing. In oneembodiment, targeted sequencing (e.g., by a combination of targetenrichment and massively parallel sequencing) can be used for obtainingrepresentative and unbiased quantitative information of differentalleles within the targeted region. An example below describes targetedsequencing. Therefore, this RHDO analysis can be applied to targetedsequencing data of maternal plasma DNA to determine which maternalalleles/haplotype are/is passed onto the fetus without prior informationregarding the parental genotypes/haplotypes.

VII. DETECTION OF DE NOVO MUTATION

Some embodiments can detect a mutation that the fetus has acquired. A denovo mutation is a mutation that is not carried by the father or themother, but is produced, for example, during gametogenesis from eitherthe father or the mother or both. Such a detection has clinical utilitybecause de novo mutations play a significant role in causing a number ofgenetic diseases e.g. hemophilia A and achondroplasia.

FIG. 24 is a flowchart illustrating a method 2400 of identifying a denovo mutation in the genome of an unborn fetus of a pregnant female. Thefetus having a father and a mother being the pregnant female, and thefather having a paternal genome with two haplotypes and the motherhaving a maternal genome with two haplotypes, the method comprising:

In step 2410, a plurality of nucleic acid molecules from a biologicalsample obtained from the pregnant female are sequenced. Note the samplecontains a mixture of maternal and fetal nucleic acids.

In step 2420, a location of each of the sequenced nucleic acid moleculein the human genome is identified. In one embodiment, the mapping of thesequences can be performed by single-ended or paired-end sequencing. Inone aspect, a mapping to the human genome to find a location does notrequire an exact match of each of the nucleotides for a location to befound.

In step 2430, for each of at least a portion of the locations, amaternal sequence and a paternal sequence are determined at the locationin question. For example, if 100 locations are determined in step 2420,then the maternal and paternal genomes at these 100 locations can bedetermined. In one embodiment, the paternal sequences are determinedfrom a sample from the father as opposed to using reference haplotypesas is described above. Thus, a mutation not in a reference genome couldstill be detected. In various embodiments, the maternal sequences can beobtained from a sample that only includes maternal DNA, or can also beobtained from the biological sample, e.g., using methods describedherein.

In step 2440, a first sequence in the plurality of nucleic acidmolecules that is not present in the determined maternal or paternalsequences is identified. In one embodiment, a comparison of the firstsequence to the determined maternal or paternal sequences requires anexact match. Thus, if the match is not exact, then the first sequence isconsidered to not be present in the determined maternal or paternalsequences. In this manner, even slight de novo mutations can beidentified since a de novo mutation can be just a single nucleotidechange. In another embodiment, a certain number of DNA fragments showingthe non-maternal and non-paternal sequence are required for the sequenceto be deemed as a de novo mutation. For example, a cutoff of 3 DNAfragments could be used to determine whether a sequence, i.e. the denovo mutation, is present or not.

In step 2450, a first fractional concentration of the first sequence inthe biological sample is determined. For example, the number of DNAfragments exhibiting the first sequence could be expressed as aproportion of all DNA fragments detected from that locus.

In step 2460, a second fractional concentration of fetal nucleic acidsin the biological sample is determined using a nucleic acid moleculethat the fetus has inherited from its father, and which is present inthe paternal genome, but which is not present in the maternal genome.Such a nucleic acid molecule might contain a first allele at a locationwhere the father is homozygous and the mother is also homozygous, butfor a different allele, and thus the fetus is an obligate heterozygote.Informative loci as described above can be used to determine the nucleicacid molecule to use to determine the second fractional concentration.

In other embodiments, the second fractional concentration can bedetermined using other approaches, such as the use of PCR assays,digital PCR assays or assays based on mass spectrometry, on the Ychromosome, a panel of genetic polymorphisms, i.e. single nucleotidepolymorphisms, or insertion-deletion polymorphisms (Lun F M F et al ClinChem 2008; 54: 1664-1672). Another alternative is to use one or moregenomic loci which exhibit different DNA methylation between the fetusand mother (Poon L L M et al. Clin Chem 2002; 48: 35-41; Chan K C A etal. Clin Chem 2006; 52: 2211-2218; U.S. Pat. No. 6,927,028).

In one embodiment, the different epigenetic status is reflected bydifferent DNA methylation patterns. The different DNA methylationpatterns can involve the RAS association domain family 1A (RASSF1A) orthe holocarboxylase synthetase (biotin-(proprionyl-CoenzymeA-carboxylase (ATP-hydrolysing)) ligase (HLCS) gene. The amount of DNAfragments with the fetal-specific DNA methylation profile can beexpressed as a proportion of all DNA fragments originating from thedifferentially methylated locus.

In step 2470, the first sequence is classified as a de novo mutation ifthe first and second fractional concentrations are about the same. Anon-maternal and non-paternal sequence originating from errors in theanalysis process, e.g. sequencing errors, is a random event and has alow probability of recurrence. Therefore, multiple DNA fragmentsexhibiting the same non-maternal and non-paternal sequence at amountssimilar to the measured fractional fetal DNA concentration for thesample are likely to be a de novo mutation present in the fetal genomerather than have arisen from sequencing error. In one embodiment, acutoff value may be used to determine whether the fractionalconcentrations are the same. For example, if the concentrations arewithin a specified value of each other, then the first sequence isclassified as a de novo mutation. In various embodiments, the specifiedvalue can be 5%, 10%, or 15%.

EXAMPLES I. Example 1

To illustrate embodiments of the present invention, the following casewas analyzed. A couple, attending an obstetrics clinic for the prenataldiagnosis of beta-thalassemia, was recruited. The father was a carrierof the -CTTT 4 base-pairs deletion of codons 41/42 of the humanbeta-globin gene. The pregnant mother was a carrier of the A→G mutationat nucleotide -28 of the human beta-globin gene. Blood samples weretaken from the father and mother. For the mother, the blood sample wastaken prior to chorionic villus sampling (CVS) at 12 weeks of gestation.Following CVS, a portion was stored for the experiment. An objective ofthe experiment was to construct a genomewide genetic map or to determinethe partial or complete genomic sequence of the fetus by the massivelyparallel sequencing of maternal plasma DNA.

1. Determination of the Parental Genotypes

DNA was extracted from the buffy coats of the father and mother, and theCVS sample. These DNA samples were subjected to analysis by theAffymetrix Genome-Wide Human SNP Array 6.0 system. This system features1.8 million genetic markers, including ˜900,000 single nucleotidepolymorphisms (SNPs) and more than ˜950,000 probes for the detection ofcopy number variations. The absolute number and the percentages of SNPsshowing different genotype combinations for the father, mother and fetus(CVS) are shown in the table of FIG. 25A.

Even though the Affymetrix system was used in this example, in practice,any genotyping platform known to those of skill in the art could beused. Indeed, apart from genotyping, the buffy coat DNA of the fatherand mother can also be subjected to sequencing, either on a whole genomebasis or for selected genomic regions. Furthermore, any source ofconstitutional DNA (e.g. buccal cell DNA, hair follicle DNA, etc) fromthe father and mother could be used establishing the parental genotypes.

The CVS sample was analyzed to provide a standard for comparison withthe fetal genetic map deduced from maternal plasma analysis. Inaddition, for this experiment, the genotype of the CVS sample can alsobe used for constructing the haplotype of the mother for RHDO analysis.In this scenario, the use of the CVS genotype for such haplotypeconstruction purpose was only used for illustration purposes. In aclinical application of embodiments, the maternal haplotype can beconstructed through the analysis of other individuals in the family, forexample, a previous offspring, a sibling, the parents or other relativesof the mother. The maternal haplotypes of the chromosomal regions ofinterest can also be constructed by other methods well known to thoseskilled in the art, some of which are mentioned herein.

For selected embodiments, the haplotype of the father of the unbornfetus to be analyzed could also be determined. This information can beparticularly useful for relative haplotype dosage for chromosomalregions in which both the father and the mother are heterozygous.

2. Massively Parallel Sequencing of Maternal Plasma DNA

Plasma DNA obtained from the mother was subjected to massively parallelsequencing using the Illumina Genome Analyzer platform. Paired-endsequencing of the plasma DNA molecules was performed. Each molecule wassequenced at each end for 50 bp, thus totaling 100 bp per molecule. Thetwo ends of each sequence were aligned to the repeat-unmasked humangenome (Hg18 NCBI.36 downloaded from UCSC http://genome.ucsc.edu) usingthe SOAP2 program from the Beijing Genomics Institute at Shenzhen(soap.genomics.org.cn)(Li Ret al. Bioinformatics 2009, 25(15):1966-7)The table, FIG. 25B, lists the alignment statistics of the first 20 flowcells. Thus, with 20 flow cells, over 3.932 billion reads were alignedto the reference human genome.

3. Calculation of the Fractional Fetal DNA Concentrations

As mentioned above, the fractional concentration of fetal DNA in thematernal plasma sample can be calculated from the sequencing data. Oneway was to analyze SNPs in which the father and mother were bothhomozygous, but for different alleles from one another. For such SNPs,the fetus would be an obligate heterozygote for one paternally-inheritedand one maternally-inherited allele. In one embodiment, any of thecalculation methods described in section V may be used. In this example,calculations were performed on the cumulative data across differentpolymorphic genetic loci that fulfilled the parental genotypeconfiguration (i.e. both parents being homozygous, but for differentalleles) on different chromosomes. The fractional concentrations offetal DNA calculated for SNPs located on different chromosomes arelisted in the right-hand-most column of FIG. 26. As can be seen from thetable, the fractional concentrations determined for SNPs located ondifferent chromosomes correlate very closely with each other.

As a quality control experiment, SNPs in which the mother was homozygousand the father was heterozygous were also investigated from theAffymetrix SNP 6.0 analysis of the buffy coat samples (middle column ofFIG. 26). It can be seen that at sufficient depth of DNA sequencing, thefractional fetal DNA concentrations measured from this analysis werevery similar to those measured for SNPs in which both the father andmother were homozygous but for different alleles.

In one implementation, when near-concordance of the fractional fetal DNAconcentrations was observed from these two types of SNPs, one couldconclude that one was close to complete sequencing coverage of the fetalgenome. In one aspect, at a lesser depth of coverage, the fractionalfetal DNA concentrations measured for SNPs in which the mother washomozygous and the father was heterozygous would be higher than thosemeasured for SNPs in which both the father and mother were homozygous,but for different alleles. At such a lesser depth of coverage, theabsence of a paternally-unique allele from the sequencing results canhave two possible causes: (i) that the fetus had not inherited thisallele from the father; and/or (ii) that the fetus had inherited thisallele from the father, but then this allele was missing from thesequencing results because the depth of sequencing was not enough.

4a. Calculation of the Percentage Coverage of the Fetal Genome

Also, as mentioned above, the percentage of the fetal genome that hadbeen analyzed by sequencing of maternal plasma DNA could be determinedby looking at the subset of SNPs in which the father and mother wereboth homozygous, but for different alleles. In this family, 45,900 SNPson the Affymetrix SNP 6.0 array belonged to this subset. The percentagecoverage of the fetal genome could be deduced by analyzing the plasmaDNA sequencing data to see in what percentage of this subset of SNPscould a fetal allele be detected by sequencing.

The plot in FIG. 27A illustrates the observed percentage of SNPs in thissubset in which a fetal allele could be seen from the sequencing datafor the first 20 flow cells analyzed. Thus, a fetal allele could beobserved in 94% of such SNPs. This degree of sequencing corresponded toover 3.932 billion reads, each with 100 bp of sequences. The plot inFIG. 27B shows the coverage vs. the number of reads, instead of thenumber of flow cells. With the increase in throughput of differentsequencing platforms, it is expected that the number of flow cells orruns that would be used or required to generate these number of sequencereads or length of sequences would decrease in the future.

In some embodiments, as multiple SNPs were detected in each chromosomalregion or chromosomes, the coverage of the fetal genome could be muchlower than 94% while still providing an accurate genome mapping. Forexample, assume there are 30 informative SNPs in a chromosomal region,but a fetal allele is detected for only 20 SNPs out of the 30 SNPs.However, the chromosomal region may still be accurately identified withthe 20 SNPs. Thus, in one embodiment, equivalent accuracy can beobtained with a coverage of less than 94%.

4b. Coverage of Genetic Map of Alleles that the Fetus had Inherited fromits Father

This illustrative analysis focuses on SNP alleles in which the fatherwas heterozygous and the mother was homozygous. In this family, 131,037SNPs on the Affymetrix SNP 6.0 platform belonged to this category. Asubset of these SNPs consisted of the 65,875 SNPs in which the motherwas homozygous, while the father and the fetus were both heterozygous.With the use of 20 flow cells, the paternally-inherited alleles could beobserved in 61,875 of these SNPs, indicating a coverage of 93.9%. Thislatter percentage fitted well with the percentage coverage data deducedin the previous paragraph. The correlation between the coverage ofpaternally-inherited alleles and the number of mappable sequence readsand the number of flow cells sequences are shown in FIG. 28A and FIG.28B, respectively.

To elucidate the specificity of this approach for detecting genuinepaternally-inherited fetal alleles, the 65,162 (i.e. 131,037 — 65,875)SNPs in which the fetus had inherited alleles that were the same asthose possessed by the mother were analyzed. For such SNPs, the apparentdetection of alleles different from those possessed by the mother wouldrepresent a false-positive. Thus, amongst the 65,162 SNPs, only 3,225false-positives (4.95%) were observed when 20 flow cells were analyzed.These false-positives can be the result of sequencing errors orgenotyping errors of the father's or mother's DNA, or de novo mutationsin the fetus. The correlation between the false-positive rate and thenumber of flow cells sequenced is shown in FIG. 29A.

The false-positive rates can also be estimated by considering the subsetof SNPs which both the father and mother were homozygous and with thesame allele. The presence of any alternative allele at the particularlocus was considered to be a false-positive. These false-positives canbe the result of sequencing errors or genotyping errors of the father'sor mother's DNA, or de novo mutations in the fetus. There were 500,673SNPs in this subset. With the sequence data from 20 flow cells,false-positive results were detected in 48,396 SNPs (9.67%). Thecorrelation between false-positive rate and the number of flow cellssequenced is shown in FIG. 29B. This false-positive rate was higher thanthe estimation using the subset of SNPs which the mother and the fetuswere homozygous and the father was heterozygous. This is because, in thelatter subset of SNPs, only the presence of the paternally inheritedallele in maternal plasma is considered to be a false-positive whereas,in the former subset, any allele other than the common allele shared bythe father and mother is considered as a false-positive result.

FIG. 30 shows the coverage of the fetal-specific SNPs for differentnumber of flow cells analyzed. The SNPs that both the father and motherwere homozygous, but with different alleles, are included in thisanalysis. The X-axis is the fold coverage of the fetal-specific SNPs,and the Y-axis is the percentage of SNPs with the specified foldcoverage. With the increase in the number of flow cells being analyzed,the average number of fold coverage for the fetal-specific SNPsincreases. For example, when one flow cell was analyzed, the averagecoverage of SNPs was 0.23 fold. The average coverage increased to 4.52fold when 20 flow cells were analyzed.

5. Accuracy of a Genetic Map Inherited from its Mother

FIG. 31 shows the accuracy of Type A analysis when data from 10 flowcells were used. Section II.B describes embodiments of a Type A and TypeB analysis (also referred to as alpha and beta). The accuracy is for thecorrect determination of the haplotype that was inherited from themother. The accuracy is separately presented for each chromosome.

Using a likelihood ratio of 1,200 for SPRT analysis (Zhou W et al. NatBiotechnol 2001;19:78-81; Karoui N E et al. Statist Med2006;25:3124-33), the accuracy ranged from 96% to 100%. As shown, evenwith such a high likelihood ratio for SPRT classification, a total of2,760 segments across the genome could be classified. This degree ofresolution is sufficient for most purposes, when one considers thatmeiotic recombinations take place at the frequency of one to a lowsingle digit number per chromosome arm per generation. In addition, onecould see that all of the misclassifications could be prevented when theinterlacing approach was used (right-hand-side of FIG. 31). As describedabove, the interlacing approach uses both Type A and Type B analysis.

FIG. 32 shows the accuracy of Type B analysis when data from 10 flowcells were used. Using a likelihood ratio of 1,200 for SPRT analysis,the accuracy ranged from 94.1% to 100%. All of the misclassificationscould be prevented when the interlacing approach was used(right-hand-side of FIG. 32), as was seen in FIG. 31.

FIG. 33 shows the accuracy of Type A analysis when the data from 20 flowcells were used. Using a likelihood ratio of 1,200 for SPRT analysis andthe “two consecutive blocks” algorithm, a total of 3,780 classificationswere made and only 3 (0.1%) classifications were incorrect. FIG. 34shows the accuracy of Type B analysis when the data from 20 flow cellswere used. Using a likelihood ratio of 1,200 for SPRT analysis and the“two consecutive blocks” algorithm, a total of 3,355 classificationswere made and only 6 (0.2%) classifications were incorrect. In theseexamples, the SPRT is performed across a number of genetic markers, suchas SNPs.

II. PRENATAL DETERMINATION OF RISK OF BETA-THALASSEMIA

In one embodiment, to determine the risk of the fetus in havingbeta-thalassemia (an autosomal recessive disease) one can determine ifthe fetus has inherited mutant alleles carried by its father and mother.In this case mentioned above, the father is a carrier of the -CTTT 4base-pairs deletion of codons 41/42 of the human beta-globin gene. Thepregnant mother was a carrier of the A♯G mutation at nucleotide -28 ofthe human beta-globin gene.

To determine if the fetus has inherited the paternal codons 41/42mutation, the sequencing data of the maternal plasma DNA, using thefirst 10 flow cells, were searched for this mutation. A total of 10reads with this mutation were found (FIG. 35A). Hence, the fetus hadinherited the paternal mutation. In addition, 62 reads were found tocontain the wildtype sequence at codons 41/42 (FIG. 35B). Thus, thepercentage of the reads in this region containing the mutation is0.1389. This figure is very close to the fractional fetal DNAconcentration determined in FIG. 26. In one embodiment, the risk of thefetus in inheriting the paternal mutation can also be determined byelucidating its inheritance of genetic polymorphisms linked to thepaternal mutation.

In one embodiment, to determine the risk that the fetus has inheritedthe maternal—28 mutation, RHDO analysis was performed. In this family,the -28 mutation was located on haplotype IV while the wildtype allelewas located on haplotype III. The results of the Type A RHDO analysisare shown in FIG. 36 while those of the Type B RHDO analysis are shownin FIG. 37. In both types of analysis, the fetal inheritance ofhaplotype III from the mother was deduced. In other words, the fetus hadinherited the wildtype allele from the mother. The final diagnosis ofthe fetus was that it has inherited the codons 41/42 mutation from thefather and a wildtype allele from the mother. Thus, the fetus is aheterozygous carrier of beta-thalassemia and thus should be clinicallyhealthy.

III. TARGET-ENRICHMENT AND TARGETED SEQUENCING

As discussed in the previous sections, the accuracy of the estimation ofthe fractional fetal DNA concentration and the resolution of the geneticmap deduced from the analysis of maternal plasma DNA can depend on thedepth of coverage of the loci-of-interest. For example, we havedemonstrated that a total of 200 molecules corresponding to a SNP locusmight be required to determine, with high accuracy, the fractional fetalDNA concentration without prior information of the maternal genotype.The allele counts for a SNP in maternal plasma can be obtained by, forexample but not limited to, real-time PCR, digital PCR and massivelyparallel sequencing.

As massively parallel sequencing of maternal plasma DNA cansimultaneously determine the allele counts for millions of SNPs acrossthe whole genome, it is an ideal platform for genomewide analysis acrossdifferent loci. The basic format of massively parallel sequencing allowsdifferent regions within the genome to be covered at similar depths.However, in order to sequence a particular region-of-interest at highsequencing depth using random massively parallel sequencing, theremaining parts of the genome (not intended to be analyzed) has to besequenced to the same extent. Thus, this approach could be costly. Toimprove the cost-effectiveness of the massively parallel sequencingapproach, one way is to enrich the target region before proceeding tosequencing. Targeted sequencing can be performed by solution phasecapture (Gnirke A, et al. Solution hybrid selection with ultra-longoligonucleotides for massively parallel targeted sequencing. NatBiotechnol 2009;27:182-9), microarray capture (e.g. using the NimbleGenplatform) or targeted amplification (Tewhey R, et al. Microdroplet-basedPCR enrichment for large-scale targeted sequencing. Nat Biotechnol2009;27:1025-31).

Targeted sequencing was initially applied to detect population geneticvariations, e.g. for genetic association studies. Therefore, its currentapplication in genomics research is aimed at solving qualitativeproblems (e.g. genotyping or mutation detection). However, theapplication of targeted sequencing in maternal plasma DNA fornoninvasive prenatal diagnosis purpose involves quantitativeconsiderations, the feasibility of which had been unclear. For example,the use of targeted sequencing might introduce quantitative bias in thedetection of fetal and maternal DNA in maternal plasma. In addition,previous work has shown that fetal DNA is shorter than maternal DNA(Chan K C A et al. Size distributions of maternal and fetal DNA inmaternal plasma. Clin Chem 2004; 50: 88-92). This size difference mightalso introduce quantitative bias or differential efficiency in thecapture of fetal and maternal DNA in maternal plasma. One was also notsure about the efficiency whereby such fragmented DNA molecules might becaptured. In the following descriptions, we demonstrate that targetedsequencing can be achieved by target enrichment followed by massivelyparallel sequencing. We also show that target enrichment is an efficientway of estimating the fractional fetal DNA concentration compared withwhole-genome sequencing.

A. Determining Fractional Concentration Using Target-Enrichment

1. Materials and Methods

Four (M6011, M6028, M6029 and M6043) pregnant women with singletonfemale fetuses were recruited. Maternal peripheral blood samples werecollected into EDTA blood tubes before elective cesarean section in thethird trimester, while placenta samples were collected after electivecesarean section. After centrifugation, DNA from the peripheral bloodcells was extracted using the Blood Mini Kit (Qiagen). DNA from 2.4 mLof plasma was extracted by the DSP DNA Blood Mini Kit (Qiagen). Maternalgenomic DNA was extracted from buffy coat and fetal genomic DNA wasextracted from placental tissues. Third trimester samples were used inthis example for illustration purposes only. First and second trimestersamples can equally be used.

Maternal and fetal genotypes were determined by the Genome-Wide HumanSNP Array 6.0 (Affymetrix). 5˜30 ng plasma DNA for each case was usedfor DNA library construction by the paired-end sample preparation kit(Illumina) according to the manufacturer's protocol of ChromatinImmunoprecipitation Sequencing sample preparation. The adapter-ligatedDNA was purified directly using spin columns provided in a QIAquick PCRpurification kit (Qiagen), without further size selection. Theadapter-ligated DNA was then amplified using a 15-cycle PCR withstandard primers. The primers were PCR Primer PE 1.0 and 2.0 fromIllumina. The DNA libraries were quantified by using a NanoDrop ND-1000spectrophotometer (NanoDrop Technologies) and run on a 2100 Bioanalyzer,using a DNA 1000 kit (Agilent), to check for size distribution. 0.6-1 μgof an amplified plasma DNA library was generated for each sample in anaverage size of about 290 bp. The capture library was obtained fromAgilent and covered 85% of the exons on the human chrX (catalog number:5190-1993). For all four cases in this study, 500 ng of the amplifiedplasma DNA library of each case was incubated with the capture probesfor 24 hours at 65° C., according to the manufacturer's instruction.After hybridization, the captured targets were selected by pulling downthe biotinylated probe/target hybrids by using streptavidin-coatedmagnetic beads (Dynal DynaMag-2 Invitrogen), and purified with theMinElute PCR Purification Kit (Qiagen). Finally, the targeted DNAlibraries were enriched by 12-cycle PCR amplification with SureSelect GAPE primers from Agilent. The PCR products were purified by QIAquick PCRPurification Kit (Qiagen). The DNA libraries prepared with or withouttarget enrichment were then subjected to random massively parallelsequencing using the Illumina Genome Analyzer IIx. One sequencing laneon a standard flow cell was used to sequence each DNA library.

2. Fractional Concentration of Fetal DNA Without Target Enrichment

The fractional fetal DNA concentration can be calculated based on theallele counts of the informative SNPs (i.e. SNPs that the mother ishomozygous and the fetus is heterozygous). The table below shows that120184, 110730, 107362 and 110321 informative SNPs were identifiedthroughout the whole genome for the four cases, while 63, 61, 69 and 65(respectively in the same case order) fell within the targeted region onchromosome X. Without target enrichment, the fractional fetal DNAconcentrations were 33.4%, 31.3%, 29.2% and 34.4% based on the data ofall informative SNPs in the genome.

Fetal Whole genome Shared specific Fractional Target informative alleleallele fetal DNA Sample enrichment SNP no. counts counts concentrationM6011 No 120,184 15,309 3,064 33.4% M6028 No 110,730 16,778 3,114 31.3%M6029 No 107,362 19,889 3,404 29.2% M6043 No 110,321 21,070 4,369 34.4%

3. Comparison of Samples With and Without Target Enrichment

In some embodiments, the depth of sequence coverage represented theaverage number of times each base had been sequenced in a particularregion. In this embodiment, we calculated the sequence depth of thetargeted region by dividing the total number of sequenced bases withinthe targeted region by the targeted region length (3.05 Mb). For theregions covered by the enrichment kit, the mean sequence coverage was0.19 times for the non-enriched samples and 54.9 times for the enrichedsamples, indicating a mean of 289-fold enrichment. At this sequencingdepth, only 4.0% of the fetal-specific alleles within the targetedregion were detected before target enrichment (see table below). Incomparison, 95.8% of them became detectable after target enrichment (seetable below). Therefore, target enrichment greatly increased thedetection rate of fetal specific alleles within the targeted region.

Then, we compared the fractional fetal DNA concentrations based on theread counts of all informative SNPs within the targeted region for eachsample, with and without enrichment. Without target enrichment, thenumber of fetal-specific reads ranged from 0 to 6 for the four samples(see table below). Due to the low sequence coverage, inadequate samplingof the fetal DNA molecules would prevent an accurate estimation of thefractional fetal DNA concentration. With target enrichment, a muchlarger number of fetal specific allele counts (511˜776) and sharedallele counts (2570˜3922) within the targeted region were observed (seetable below). The fetal DNA percentages were calculated as 35.4%, 33.2%,26.1% and 33.0%, consistent with the fetal DNA percentages estimated bythe genomewide data in the non-enriched samples (see table below). Theseresults indicated that maternal and fetal DNA molecules were enriched toa similar extent within the targeted region.

No. of informative No. of Fetal SNP detectable specific Fetal within thefetal allele Shared specific Fractional Target targeted specificdetection allele allele fetal DNA Sample enrichment region alleles ratecounts counts concentration M6011 No 63  6  9.5%  13  6 63.2% M6028 No61  2  3.3%   6  2 50.0% M6029 No 69  2  2.9%  11  2 30.8% M6043 No 65 0  0.0%  15  0  0.0% M6011 Yes 63 60 95.2% 3072 661 35.4% M6028 Yes 6160 98.4% 2570 511 33.2% M6029 Yes 69 66 95.7% 3835 575 26.1% M6034 Yes65 61 93.9% 3922 776 33.0%

B. Determining Fetal Genome Using Target-Enrichment

One application of an RHDO method is for the noninvasive prenataldetection of maternally inherited genetic diseases. Using massivelyparallel sequencing of maternal plasma without target enrichment, RHDOanalysis can accurately determine which maternal haplotype is passedonto the fetus with an average of 17 SNPs when the sequencing depth ofmaternal plasma DNA is approximately 65-fold human genome coverage. Toimprove the cost-effectiveness of this approach, selectively directingthe sequencing to specific regions of interest within the genome and tothen applying an RHDO analysis to the sequencing data can be performed.As an example, we demonstrated the concept using the targeted sequencingand RHDO analysis of chromosome X. However, the targeted sequencing andRHDO analysis can also be applied to all chromosomes, e.g. theautosomes. In one embodiment, an RHDO analysis as described above can beused for the targeted embodiments.

Five (PW226, PW263, PW316, PW370 and PW421) pregnant women withsingleton male fetuses were recruited. Maternal peripheral blood sampleswere collected into EDTA blood tubes before chorionic villus sampling inthe first trimester. After centrifugation, DNA from the peripheral bloodcells was extracted using the Blood Mini Kit (Qiagen). DNA from 3.2 mLof plasma was extracted by the DSP DNA Blood Mini Kit (Qiagen). Maternalgenomic DNA was extracted from the buffy coat and fetal genomic DNA wasextracted from the chorionic villi. The samples were prepared andanalyzed as described above. Each sample was then sequenced randomlyusing one lane on an Illumina flow cell.

In this example, we used the fetal genotype, along with sequencinginformation from nucleic acids of the mother, to deduce the maternalhaplotypes for chromosome X and deduce which haplotype was inheritedfrom the mother. For each SNP on chromosome X that the mother washeterozygous (i.e., an informative SNP), the allele that was inheritedby the fetus is defined as coming from the maternal haplotype 1 (Hap I)whereas the maternal allele that was not passed onto the fetus wasdefined as coming from the maternal haplotype 2 (Hap II). In someembodiments, for clinical applications, the fetal genotype may not beavailable beforehand and the maternal haplotypes can be determined orinferred by methods well-known to those skilled in the art and methodsdescribed herein. Chromosome X is used here for illustration purposesonly. Other chromosomes, e.g. the autosomes, can also be used in suchanalysis.

For the five cases described here, all of them were carrying a singletonmale fetus. As a male fetus only inherits one chromosome X from themother but no chromosome X from the father, the maternal chromosome Xthat was passed onto the fetus would be overrepresented in the maternalplasma. The RHDO analysis was carried out from the pter to qter ofchromosome X. Starting with the SNP closest to the pter of chromosome X,SPRT analysis can determine if the allele on Hap I or Hap II wasstatistically significantly overrepresented in the maternal plasma. Ifnone of the two haplotypes was statistically significantlyoverrepresented, the allelic counts for the next SNP can be combined forfurther SPRT analysis. Additional SNPs can be combined for analysisuntil the SPRT process identified one of the haplotypes as statisticallysignificantly overrepresented. The classification process can then berestarted at the next SNP.

FIGS. 38A and 38B shows the SPRT classification results for case PW226as an example. There were a total of nine successful SPRTclassifications for chromosome X in this case. For each SPRTclassification, the alleles on Hap I was shown to be overrepresented inthe maternal plasma sample, indicating that the fetus had inherited HapI from the mother. As we defined Hap Ito be the haplotype containing thealleles passed onto the fetus, the results of all these SPRTclassification were correct.

The RHDO analysis results for the five cases are summarized in FIG. 39.The number of successful SPRT classifications ranged from 1 to 9. All ofthe SPRT classifications were correct. A higher fractional fetal DNAconcentration was associated with a higher number of classifications.This is because the allelic imbalance due to the presence of fetal DNAcan be detected more easily when the fractional concentration of fetalDNA is higher. Therefore, fewer SNPs may be needed to reach a successfulRHDO classification. Defined chromosomal region(s) can thus be dividedinto more RHDO blocks. Our results confirm that RHDO analysis can beperformed on the massively sequencing data which are obtained aftertarget enrichment.

Our data further showed that the targeted approach is a morecost-effective way of performing RHDO analysis. Without targetenrichment, for samples with similar fetal DNA concentrations,sequencing by approximately 5 flow cells (i.e. 40 sequencing lanes) wasrequired (FIG. 40) to reach the average depth achieved for samples shownin FIG. 39. Here we show that with target enrichment, sequencing by onlyone lane already reaches the average sequencing depth of some 15 to 19fold for successful RHDO classification. Alternatively, even higherfold-level of sequencing coverage could be achieved with relativelylittle additional cost when target enrichment is used. The higher levelof sequencing coverage can effectively reduce the size of the genomicregion required for successful RHDO classification and hence improvesthe resolution of the analysis.

IV. TARGET-ENRICHMENT

It has been known since 2004 that circulating fetal DNA molecules aregenerally shorter than maternal DNA in maternal plasma (Chan K C A et alClin Chem 2004; 50: 88-92; Li et al Clin Chem 2004). However, themolecular basis of this observation remained unsolved. In our currentstudy, we generated 3.931×10⁹ reads in the study plasma sample and used1-bp bins in our bioinformatics analysis. The size of each sequencedplasma DNA molecule were deduced from the genome coordinates of the endsof the paired-end reads.

For this analysis, we focused on single nucleotide polymorphisms (SNPs)in which the father and mother were both homozygous, but for a differentallele. For such SNPs, the fetus was an obligate heterozygote. Theallele for each SNP that the fetus had inherited from the father couldbe used as a fetal-specific marker. The sizes of the fetal (using thepaternally-inherited fetal-specific alleles) and total sequences weredetermined for the whole genome (FIG. 41) and individually for eachchromosome (FIG. 42A-42C).

We observed that the most significant differences between fetal andmaternal DNA in maternal plasma is the reduction in the 166 bp peak,relative to the 143 bp peak (FIG. 41). The most abundant total sequences(predominantly maternal) were 166 bp in length. The most significantdifference in the size distribution between the fetal and total DNA wasthat fetal DNA exhibited a reduction in the 166 bp peak (FIG. 41) and arelative prominence of the 143 bp peak. The latter likely correspondedto the trimming of a ˜20-bp linker fragment from a nucleosome to itscore particle of ˜146 bp (Lewin B, in Gene I X, Jones and Bartlett,Sudbury, 2008, pp. 757-795).

From approximately 143 bp and below, the distributions of both fetal andtotal DNA demonstrated a 10 bp periodicity reminiscent ofnuclease-cleaved nucleosomes. These data suggest that plasma DNAfragments are derived from apoptotic enzymatic processing. In contrast,size analysis of reads that mapped to the non-histone boundmitochondrial genome did not show this nucleosomal pattern (FIG. 41).These results provide a previously unknown molecular explanation for theknown size differences between fetal and maternal DNA using Y chromosomeand selected polymorphic genetic markers (Chan K C A et al Clin Chem2004; 50: 88-92; Li et al Clin Chem 2004; 50: 1002-1011; US PatentApplication 20050164241; US Patent application 20070202525), and showthat such size differences exist across the entire genome. The mostlikely explanation of this difference is that circulating fetal DNAmolecules consist of more molecules in which the ˜20 bp linker fragmenthas been trimmed from a nucleosome.

Given these observations, there are a number of ways in which the samplecan be enriched for fetal DNA. In one embodiment, one can use reagentsthat would preferentially bind to the linker fragment. Such reagentswould be expected to bind preferentially to maternal-derived DNA whencompared with fetal-derived DNA in maternal plasma. One example of suchreagents is an antibody. One target of such an antibody is one thatbinds to histone H1. Histone H1 is known to bind to the linker fragment.One application of such an antibody is for performing enrichment offetal DNA by negative selection, i.e., via the preferentialimmunoprecipitation of the maternally-derived DNA in maternal plasmathat contains the linker, histone H1-containing, fragment. Furthermore,H1 is known to have a number of variants, some of them exhibitingtissue-specific variation in expression (Sancho M et al PLoS Genet 2008;4: e1000227). These variants might be further exploited to differentiatethe fetal (predominantly placental) and maternal (predominantlyhematopoietic (Lui Y Y N et al Clin Chem 2002; 48: 421-427) DNA. Forexample, one can target a histone H1 variant that is predominantlyexpressed by trophoblastic cells to preferentially and positively selectfor fetal-derived DNA in maternal plasma. This strategy can also beapplied for other histone proteins or other nucleosomal proteins thatexhibit tissue-specific, especially trophoblast-specific, patterns ofexpression.

Given the sharp 166 bp peak for maternal DNA, another possibility forenriching fetal DNA is to design a system for negative selection of DNAfragments that are of 166±2 bp in length. For example, a system based oncapillary electrophoresis or high performance liquid chromatographycould allow precise size measurement and separation of DNA molecules.Another way for negative selection is to do this in silico during thebioinformatic analysis of the sequencing data.

As other DNA species in plasma, e.g. tumor DNA (Vlassov V V et al. CurrMol Med 2010; 10: 142-165) and transplanted organ DNA (Lo Y M D et alLancet 1998; 351: 1329-1330), is also expected to share such featureswith fetal DNA in maternal plasma, the strategies listed in (1) and (2)above could also be used for the enrichment of these DNA species.

According to one embodiment, a method for the differential enrichment ofDNA species in human plasma or serum through the targeting of the linkerfragment of the nucleosomes is provided. In an embodiment, theenrichment is made by removing one of the following: maternally-derivedDNA or DNA derived from hematopoietic cells. In another embodiment, thetargeting involves a reagent (such as an antibody or another type ofprotein) that would bind preferentially to a protein or nucleic acidcomponent of the linker fragment of the nucleosome. In anotherembodiment, the targeting reagent will selectively bind to histone H1 oranother protein that binds to the linker fragment of the nucleosome. Inanother embodiment, the targeting reagent will bind to maternal orhematological variants of histone H1 or another protein that binds tothe linker fragment of the nucleosome. In one embodiment, the removal ofthe DNA is carried out by immunoprecipitation or binding to a solidsurface.

According to another embodiment, a method for the differentialenrichment of fetal DNA in maternal plasma or serum includes: (a) use ofan antibody that would bind to one or more components of the linkerfragment of the nucleosome; (b) remove the bound fraction byimmunoprecipitation or capture to a solid surface; and (c) harvest theunbound fraction which contains an increased fractional concentration offetal DNA.

Any of the software components or functions described in thisapplication, may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a computer readable medium for storage and/ortransmission, suitable media include random access memory (RAM), a readonly memory (ROM), a magnetic medium such as a hard-drive or a floppydisk, or an optical medium such as a compact disk (CD) or DVD (digitalversatile disk), flash memory, and the like. The computer readablemedium may be any combination of such storage or transmission devices.

Such programs may also be encoded and transmitted using carrier signalsadapted for transmission via wired, optical, and/or wireless networksconforming to a variety of protocols, including the Internet. As such, acomputer readable medium according to an embodiment of the presentinvention may be created using a data signal encoded with such programs.Computer readable media encoded with the program code may be packagedwith a compatible device or provided separately from other devices(e.g., via Internet download). Any such computer readable medium mayreside on or within a single computer program product (e.g. a hard driveor an entire computer system), and may be present on or within differentcomputer program products within a system or network. A computer systemmay include a monitor, printer, or other suitable display for providingany of the results mentioned herein to a user.

An example of a computer system is shown in FIG. 43. The subsystemsshown in FIG. 43 are interconnected via a system bus 4375. Additionalsubsystems such as a printer 4374, keyboard 4378, fixed disk 4379,monitor 4376, which is coupled to display adapter 4382, and others areshown. Peripherals and input/output (I/O) devices, which couple to I/Ocontroller 4371, can be connected to the computer system by any numberof means known in the art, such as serial port 4377. For example, serialport 4377 or external interface 4381 can be used to connect the computerapparatus to a wide area network such as the Internet, a mouse inputdevice, or a scanner. The interconnection via system bus allows thecentral processor 4373 to communicate with each subsystem and to controlthe execution of instructions from system memory 4372 or the fixed disk4379, as well as the exchange of information between subsystems. Thesystem memory 4372 and/or the fixed disk 4379 may embody a computerreadable medium. Any of the values mentioned herein can be output fromone component to another component and can be output to the user.

A computer system can include a plurality of the same components orsubsystems, e.g., connected together by external interface 4381 or by aninternal interface. In some embodiments, computer systems, subsystem, orapparatuses can communicate over a network. In such instances, onecomputer can be considered a client and another computer a server, whereeach can be part of a same computer system. A client and a server caneach include multiple systems, subsystems, or components.

The specific details of particular embodiments may be combined in anysuitable manner or varied from those shown and described herein withoutdeparting from the spirit and scope of embodiments of the invention.

The above description of exemplary embodiments of the invention has beenpresented for the purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdescribed, and many modifications and variations are possible in lightof the teaching above. The embodiments were chosen and described inorder to best explain the principles of the invention and its practicalapplications to thereby enable others skilled in the art to best utilizethe invention in various embodiments and with various modifications asare suited to the particular use contemplated.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method of determining a fractionalconcentration of fetal DNA in a biological sample taken from a femalepregnant with a fetus, the fetus having a father and a mother being thepregnant female, wherein the biological sample contains a mixture ofmaternal and fetal nucleic acids, the method comprising: enriching thebiological sample obtained from the pregnant female for nucleic acidmolecules in a target region; sequencing a plurality of nucleic acidmolecules from the enriched biological sample to obtain sequencingresults, the sequencing being specific to the target region, wherein thesequencing results are analyzed to: identify a location of the nucleicacid molecule in the target region of the human genome; and determine arespective allele of the nucleic acid molecule; determining one or morefirst loci, wherein the fetal genome is heterozygous at each first locisuch that the fetal genome has a respective first and second allele atthat first loci, and wherein a maternal genome is homozygous at eachfirst loci such that the maternal genome has two of the respectivesecond allele at that first loci, the first allele being different thanthe second allele; for at least one of the first loci: determining afirst number P of counts of the respective first allele and a secondnumber Q of counts of the respective second allele; and determining thefractional concentration based on the first and second numbers.
 2. Themethod of claim 1, wherein determining the one or more first lociincludes: determining the paternal genome is homozygous for therespective first allele at each loci of the one or more first loci anddetermining the maternal genome is homozygous for the respective secondallele at the same locus.
 3. The method of claim 1, wherein determininga specific locus to be the one or more first loci includes: determininga cutoff value for a number of predicted counts of the respective firstallele at the specific locus, the cutoff value predicting whether thematernal genome is homozygous and the fetal genome is heterozygous,wherein the cutoff value is determine based on a statisticaldistribution of numbers of counts for different combinations ofhomozygosity and heterozygosity at the specific locus; based on theanalysis of the sequencing results, detecting the respective first andsecond allele at the specific locus; determining a number of actualcounts of the respective first allele based on the sequencing of theplurality of nucleic acid molecules from the biological sample; anddetermining the specific locus is one of the first loci when the numberof actual counts is less than the cutoff value.
 4. The method of claim3, wherein determining the cutoff value includes determining astatistical distribution for a maximum and a minimum fractionalconcentration.
 5. The method of claim 4, wherein the statisticaldistribution is a Poisson distribution.
 6. The method of claim 4,wherein the maximum and the minimum fractional concentration depend onthe length of the pregnancy.
 7. The method of claim 1, wherein enrichingthe biological sample comprises performing solution phase capture,microarray capture, or targeted amplification.
 8. The method of claim 1,wherein sequencing the plurality of nucleic acid molecules comprisessequencing both ends of each nucleic acid molecule of the plurality ofnucleic acid molecules.
 9. The method of claim 1, wherein sequencing theplurality of nucleic acid molecules comprises random sequencing of theenriched biological sample.
 10. The method of claim 1, furthercomprising: determining the number of molecules to be analyzed at afirst locus of the one or more first loci using an assumed value offractional concentration of fetal DNA and a target probability ofdetecting the respective first allele at the first locus.
 11. The methodof claim 1, further comprising: for a specific locus: comparing countsof the respective first allele to a cutoff value, and categorizing,using the comparison of the counts to the cutoff value, the locus as alocus where the mother is homozygous in the first allele and the fetusis heterozygous; the mother is heterozygous and the fetus isheterozygous; or the mother is heterozygous and the fetus is homozygous.12. The method of claim 1, wherein the fractional concentration isdetermined as 2×P/(P+Q).
 13. The method of claim 1, wherein P and Q aredetermined for a plurality of first loci, and where the fractionalconcentration f is determined as${f = \frac{\sum_{i = 1}^{n}{2P_{i}}}{\sum_{i = 1}^{n}\left( {P_{i} + Q_{i}} \right)}},$where p_(i) is the first number for the ith first loci and q_(i) is thesecond number for the ith first loci.
 14. A computer product comprisinga non-transitory computer readable medium storing a plurality ofinstructions that when executed control a computer system to determine afractional concentration of fetal DNA in a biological sample taken froma female pregnant with a fetus, the fetus having a father and a motherbeing the pregnant female, wherein the biological sample contains amixture of maternal and fetal nucleic acids, the instructionscomprising: receiving sequencing results of a sequencing of a pluralityof nucleic acid molecules from an enriched biological sample, theenriched biological sample being enriched for nucleic acid molecules ina target region, the sequencing being specific to the target region,wherein the sequencing results are analyzed to: identify a location ofthe nucleic acid molecule in the target region of the human genome; anddetermine a respective allele of the nucleic acid molecule; determiningone or more first loci, wherein the fetal genome is heterozygous at eachfirst loci such that the fetal genome has a respective first and secondallele at that first loci, and wherein a maternal genome is homozygousat each first loci such that the maternal genome has two of therespective second allele at that first loci, the first allele beingdifferent than the second allele; for at least one of the first loci:determining a first number P of counts of the respective first alleleand a second number Q of counts of the respective second allele; anddetermining the fractional concentration based on the first and secondnumbers.
 15. A system comprising: the computer product of claim 14; andone or more processors for executing instructions stored on the computerreadable medium.
 16. A system comprising: an enrichment systemconfigured to enrich a biological sample for nucleic acid molecules in atarget region to form an enriched biological sample; a sequencing deviceconfigured to sequence a plurality of nucleic molecules in the enrichedbiological sample; and a computer system comprising: a computer productcomprising a non-transitory computer readable medium storing a pluralityof instructions that when executed control a computer system todetermine a fractional concentration of fetal DNA in the biologicalsample taken from a female pregnant with a fetus, the fetus having afather and a mother being the pregnant female, wherein the biologicalsample contains a mixture of maternal and fetal nucleic acids, theinstructions comprising: receiving sequencing results from thesequencing device of a sequencing of a plurality of nucleic acidmolecules from the enriched biological sample from the enrichmentsystem, the enriched biological sample being enriched for nucleic acidmolecules in the target region, the sequencing being specific to thetarget region, wherein the sequencing results are analyzed to: identifya location of the nucleic acid molecule in the target region of thehuman genome; and determine a respective allele of the nucleic acidmolecule; determining one or more first loci, wherein the fetal genomeis heterozygous at each first loci such that the fetal genome has arespective first and second allele at that first loci, and wherein amaternal genome is homozygous at each first loci such that the maternalgenome has two of the respective second allele at that first loci, thefirst allele being different than the second allele; for at least one ofthe first loci: determining a first number P of counts of the respectivefirst allele and a second number Q of counts of the respective secondallele; and determining the fractional concentration based on the firstand second numbers; and one or more processors for executinginstructions stored on the computer readable medium.